(Note: The preparation of the last three items is described in Question 11).

PROCEDURE:

1. Grow 3 ml overnight cultures of plasmid-containing bacteria.

2. After chilling overnight cultures on ice for 5 minutes, collect the bacteria in 1.5 ml microcentrifuge tubes by centrifugation. After decanting, centrifuge the tubes 10 seconds and aspirate the remaining liquid.

3. Resuspend each pellet in 0.1 ml GET.

4. Add 2 microliters RNase A, 10 mg/ml. Mix. Allow to sit on ice for 30 minutes.

5. Add 0.2 ml NaOH/SDS. Mix with three sharp inversions. Incubate on ice 10 min. The solution will become milky as SDS precipitates.

6. Add 0.15 ml 7.5 M room teperature ammonium acetate. Mix with three sharp inversions. The precipitate will become curd-like. Continue to incubate on ice for at least 30 min.

7. Centrifuge 5 minutes.

8. Aspirate into clean microcentrifuge tubes, trying to avoid most of the precipitate. Recentrifuge for 3 minutes and again aspirate to clean tubes.

9. (Optional. See Notes.) Add 2 microliters RNase A, 10 mg/ml, and mix. Incubate at 37 deg C for 30 minutes.

10. Mix silica slurry with saturated NaI/saturated Na2SO3, using 5 microliters of silica suspension per ml NaI/Na2SO3. Before the silica settles, add 0.9 ml to each sample and mix gently. Put samples on ice for at least 1 hr.

11. Centrifuge the samples 5 minutes and decant.

12. Wash the silica pellet three times with 0.2 ml NEET solution, Vortexing each time to suspend and then centrifuging one minute. After the last wash is decanted, centrifuge 10 seconds and aspirate the remaining liquid.

13. Add 20 microliters 10 mM Tris-HCl (pH 7.5-8)/1 mM EDTA (TE) to each sample, Vortex to suspend, and incubate at 45-55 degrees C for 3 minutes. Centrifuge one minute; aspirate to clean tubes.

14. Repeat the step above, giving a total of about 40 microliters supernatent for each sample. Centrifuge three minutes and again aspirate to clean microcentrifuge tubes.

15. Remove one microliter of each sample for agarose electrophoresis.

16. If electrophoresis indicates an adequate yield, the samples can be denatured according to the Sequenase protocol, using twice the recommended volumes: Add four microliters 2 N NaOH, mix, and incubate at room temperature 5 minutes. Add 16 microliters 5 M ammonium acetate and 0.2 ml EtOH. Mix, chill the samples, collect by centrifugation, wash with 0.4 ml 70% EtOH at room temperature, and dry. Dissolve in 7 microliters H2O or TE and proceed with sequencing.

1. Use two successive centrifugations to collect bacteria from 3 ml in a single tube.

2. Lysozyme may be needed at this step for some bacterial strains. It is not needed for E. coli strains AB1157, DH1, or HB101.

3. Vigorous mixing at these steps is not only unnecessary, but leads to breakage for bacterial DNA, with subsequent contamination of the plasmid. Three sharp snaps of the wrist is adequate. (The mixture will not appear uniform -- do not worry about it.)

4. It is easier to perform a second centrifugation to remove any precipitate that is carried over than it is to avoid the precipitate completely.

5. RNA digestion during the first incubation is variable, so a second RNase treatment is sometimes needed. An alternative would be to treat with RNase just before denaturation if gel electrophoresis shows it to be necessary; RNase added before the denaturation step does not interfere with Sequenase sequencing. If RNA is BARELY visible on the agarose gel, it doesn't seem to seriously interfere with the sequencing.

6. If the NaI solution is added to the plasmid preparation first, followed by the glassmilk, the silica clumps and is difficult to resuspend. Mixing the NaI and glassmilk and then adding the mixture to the preparation avoids this difficulty.

7. As noted earlier, it is easier to remove solid material carried over during the aspiration than to avoid it. If the silica contains very fine particles, there will be some carry-over even after the second centrifugation, but small amounts do not interfere with Sequenase sequencing.

8. The samples can be stored in the freezer, either in the native state or after denaturation, and either dry or redissolved.

   The preparations appear to be dirtier than those produced by other protocols; in addition to covalently closed circular plasmid, there are open circles, denatured plasmid, and a small amount of linear plasmid, plus chromosomal DNA and, sometimes, a small amount of RNA. Nonetheless, the sequences obtained are strong and unambiguous. The improved sequencing results may be due at least in part to improved yields with this method. This method has been used to prepare pBR322 for both Sequenase double-strand sequencing and the PRISM dye terminator cycle sequencing system of ABI; The denaturation step is not needed for cycle sequencing.

14. Is there a simple subcloning method for plasmid construction?

Cut your vector and insert fragments and run them out on a low melting agarose gel (eg. SeaPlaque from FMC BioProducts) in TAE buffer. Excise the bands of interest on a LONG WAVE UV box making sure to cut the smallest slice possible. For very low percentage agarose, use a thin layer of standard agaorse (1%) as a support by pouring it without a comb, setting the comb higher, and pouring your low melting agarose gel on top once the support layer has set. Cut this layer off as well when you excise your bands. Place the gel slices in sterile eppendorfs and melt them at 70C in a beaker. Do not add any buffer or dilute the slices. Carry this beaker to the -70 freezer and put the samples in an isopropanol bath. Wait 5 minutes, and then thaw them. Spin 5 minutes in the microfuge. The supenatant is your sample for ligation. Take 2 ul of vector supernatant and distribute it to 4 screw top eppendorf tubes. Add 3 ul of BRL ligase buffer (5X w/ PEG), 8 U of NEB ligase for sticky ends, and either 1, 3, or 7 ul of insert supernatant. Bring the volume to 15 ul with sterile water. Incubate at 16 C overnight by emersing the entire tube beneath the suface of a water bath in the cold room. In the morning dilute your 15 ul ligation 5X to 75 ul and add 20 ul per 250 ul of Ca/Rb competent cells.

Slice both fragments from the gel and put them together in a tube. Bind the DNA fragments to glass-milk, wash 3x with the NEET buffer. After the final spin, dry the glass-milk DNA complex and then add 10 ul of water. Put the tube at 45-50 C for 5 minutes, then flick the tube a few times. Incubate another 5 minutes and then spin out the glass-milk. Put on ice until you remove the DNA for ligation. (you lose 3 ul due to the glass-milk). Use 7 ul mixed DNA plus 2 ul 5x ligase buffer plus 1 ul of T4 DNA ligase on ice _OR_ simply add an equal volume of 2x ligation buffer and 0.5-1.0 ul of ligase. Place at 12-16 C overnight. Transform 100 ul of competent cells with the entire amount of DNA and plate onto selective media.

Transformation:

There are many methods which will give you a number of transformants when you only need to change hosts or if you recieve plasmid DNA from someone else. A simple technique is to centrifuge exponentially growing (~2 x 10^8 cfu/ml) cells at 3000 rpm and then resuspend them in 1:20 volume of cold (4 C) 75mM CaCl2. Generally, 50-100 ul of concentrated cells are placed in a microcentrifuge tube. Keep the cells on ice with DNA added, heat shock at 42 C for 90-120 seconds, add fresh broth to express for about an hour at 37 C. Plate the undiluted mixture or a microfuge concentrated portion directly onto dry, selective media.

For "ultra-competent" E. coli cells, try the method of Inoue1990 which is the method preferred by netters:

1. Inoculate from an overnight grown in LB
2. Grow in 250 ml "SOB" at 18 degrees C until OD₆₀₀ = 0.6
3. On ice for 10 minutes.
4. Spin at 2500 x g (3000 rpm in a Beckman J-6B centrifuge) for 10 min. at 4C.
5. Resuspend cells gently in 80 ml of ice cold "TB".
6. On ice for 10 minutes.
7. Spin at 2500 x g (3000 rpm in a Beckman J-6B centrifuge) for 10 min. at 4C.
8. Resuspend cells gently in 20 ml of ice cold "TB"
9. Add DMSO to a final concentration of 7%.
10. On ice for 10 minutes.
11. Aliquot into 1-2 ml and freeze in liquid nitrogen.
12. Store frozen in liquid nitrogen.

"TB":
10 mM Pipes
55 mM MnCl2
15 mM CaCl2
250 mM KCl
pH 6.7


"SOB":
2% (w/v) bacto tryptone
0.5% (w/v) yeast extract
10 mM NaCl
2.5 mM KCl
10 mM MgCl2
10 mM MgSO4
pH 7.0

Electroporation:

Electroporation of E. coli is well established in the scientific literature, where transformation efficiencies of 10¹⁰ transformants per ug of supercoiled plasmid DNA can be expected for several E. coli strains. The advantages of electroporation over chemical methods include ease and speed. Many people have used the following protocol for High Efficiency Electro-transformation of E. coli :

1. Preparation of Cells:

1. Inoculate 1 liter of L-broth(a) with 1/100 volume of a fresh overnight culture.

2. Grow cells at 37 C with vigorous shaking to an A₆₀₀ of approximately 0.5-0.7 which should give approx. 1 x 10⁸ CFU/ml (the best results are obtained with cells that are harvested at early- to mid-log phase; the appropriate cell density therefore depends on the strain and growth conditions). In general, this will be reached 2.5 to 3 hours after diluting an overnight culture 1:50.

3. To harvest, centrifuge cells in cold centrifuge bottles in a cold rotor at 4000 x g for 15 min.

4. Remove as much of the supernatant (medium) as possible. It is better to sacrifice the yield by pouring off a few cells than to leave any supernatant behind.

5. Gently resuspend the pellets in a total of 1 liter of ice-cold 10% glycerol(b) taking care not to lyse them. Centrifuge as in step 3.

6. Resuspend in 0.5 liter of ice-cold 10% glycerol. Centrifuge as in step 3.

7. Resuspend in approximately 250 ml of ice-cold 10% glycerol. Centrifuge as in step 3.

8. Resuspend to a final volume of 3 to 4 ml in ice-cold 10% glycerol. The cell concentration should be about 1-3 x 10¹⁰ cells/ml.

9. This suspension may be frozen in aliquots on dry ice, and stored at -70 C. The cells are good for at least 6 months under these conditions.

For items 3-7 above, keep the cells as close to 0 C as possible (in an ice/water bath) throughout their preparation.

2. Electro-transformation and Plating

1. Gently thaw the cells at room temperature and then immediately place them on ice. Remove the sterile electroporation cuvettes from their pouches and place on ice.

2. In a cold, 1.5 ml polypropylene tube, mix 40 ul of the cell suspension with 1 to 2 ul of DNA (typically 10 pg/ul). [DNA should be in a low ionic strength buffer such as TE(c).] Mix well and let sit on ice about 0.5 to 1 minute.

3. Transfer the mixture of cells and DNA to a cold electroporation cuvette, making sure that the liquid is in contact with both metal faces of the cuvette.

4. Pulse once at a field strength of 18 kV/cm and a time constant of 4.5 to 5.5 milliseconds.

5. Remove the cuvette from the chamber and immediately add 1 ml of SOC(d) medium (at room temp.) to the cuvette and quickly, but gently, resuspend the cells with a pipette. (This rapid addition of SOC after the pulse is very important in maximizing the recovery of transformants.)

6. Transfer the cell suspension to a 17 x 100 mm polypropylene tube and incubate at 37 C for 1 hour. (Shaking the tubes at 225 RPM during this incubation may improve the recovery of transformants.)

7. Check and record the pulse parameters. The time constant should be close to 5 milliseconds. The field strength can be calculated as the actual volts delivered (kV) / cuvette gap (cm).

8. Plate on selective medium.

Notes:

1. L-Broth: 1% Bacto tryptone, 0.5% Bacto yeast extract, 0.5% NaCl.

2. 10% Glycerol: Prepare fresh weekly with sterilized water. Do not autoclave or filter-sterilize the glycerol solution.

3. DNA containing too much salt will make the sample too conductive and cause arcing at high voltage. TE: 10 mM Tris-HCl, 1 mM EDTA, pH 8.0.

4. SOC: 2% Bacto tryptone, 0.5% Bacto yeast extract, 10 mM NaCl, 2.5 mM KCl, 10 mM MgCl2, 10 mM MgSO4, 20 mM glucose.

Caution should be followed if you plan to re-use cuvettes (see Question 16). pertaining to re-using electroporation cuvettes). There are obvious sterility and cross-contamination risks; since electroporation is the most efficient E. coli transformation method available, even a minute amount of DNA from the previous pulse is likely to become a problem. Less obvious is the observation that electro-transformation efficiencies are highest in a uniform electric field (i.e., between two parallel electrodes). A high-voltage pulse of a high concentration of E. coli cells causes the cells in contact with the aluminum surface of the cuvette to actually "bake" onto the electrode. This obstruction of the dead cells has the same electrical effect as pitting the electrodes with either a physical "cleaning" procedure or a harsh chemical treatment of acid, or base such as bleach, in that the smooth surfaces of the electrodes are compromised. The net result is a nonuniform electric field for the pulse, and a lower transformation efficiency.

The following references deal with many aspects of electro-transformation: Chuang1995, Dower1988, Eynard1992, Hengen1995Juntibs. Jupin1995, Kubiniec1990, O'Callaghan1990, Potter1993, Sheng1995, Sixou1991, Solioz1990, Speyer1990, Steele1994, Taketo1988, Thomas1994, and Wirth1989. Additional bacterial electro-transformation references are avialable from Connie Rickey of Bio-Rad Laboratories at crickey@haley.genetics.bio-rad.com

Opinion varies concerning the life of electroporation cuvettes, but they can usually be re-used 3 to 8 times before they become unsafe.

Here are two methods which can be used for cleaning the cuvettes:

1. Simple Method:

1. Wash out cuvettes with bleach.
2. Rinse 6 times with dH2O
3. Rinse with 95% EtOH.
4. Fill again with 95% EtOH, replace cap, invert, tip off EtOH and dry upside-down in hood.

2. Paranoid Method:

1. Wash out cuvettes with bleach.
2. Rinse 6 times with dH2O.
3. Fill with 0.25 M HCl, stand at room temperature for 15 min. to 2 hours.
4. Rinse in dH2O, boil in fresh, clean, distilled water for 10 min.
5. Remove from bath, immediately rinse in 95% EtOH and dry upside down under the hood.
   Caps can be rinsed in 70% EtOH.

   Note: Do not store cuvettes for any length of time in the presence of moisture.

PCR is an acronym which stands for polymerase chain reaction . The PCR technique is basically a primer extension reaction for amplifying specific nucleic acids in vitro. The use of a thermostable polymerase allows the dissociation of newly formed complimentary DNA and subsequent annealling or hybridization of primers to the target sequence with minimal loss of enzymatic activity. PCR will allow a short stretch of DNA (usually fewer than 3000 bp) to be amplified to about a million fold so that one can determine its size, nucleotide sequence, etc. The particular stretch of DNA to be amplified, called the target sequence, is identified by a specific pair of DNA primers, oligonucleotides usually about 20 oligonucleotides in lenth. PCR has revolutionized molecular genetics and continues to be applied to many fields of biology.

• A new book "PCR Primer: A Laboratory Manual," edited by Carl Dieffenbach and Gabriela Dveksler, has been touted as being outstanding. It has a lot of practical information, from very basic to complex. This book is an excellent resource, and a good all-purpose laboratory PCR manual.

• Innis, M. A., D. H. Gelfand and J. J. Sninsky. 1995. PCR Stratagies
  Academic Press, San Diego

• Mullis, K. 1994. The Polymerase chain reaction.
  Birkhauser Press, Boston, Mass.

• Griffin, H. G. and A. M. Griffin. 1994. PCR Technology: Current Innovations
  CRC Press, Boca Raton, Florida.

• White, B. A. 1993. PCR Protocols
  Humana Press, Totowa

• Mc Pherson, M. J., P. Quirke and G.R. Taylor. 1992. PCR:
  A Practical Approach. IRL Press.

• Innis, M. A., D. H. Gelfand, J. J. Sninsky, and T. J. White. 1990.
  PCR Protocols: A guide to methods and applications. Academic Press, New York.

• Erlich, H. A. 1989. PCR technology: Principles and applications for DNA amplification.
  Stockton Press, New York.

• Ellingboe, J., and U. B. Gyllensten. 1992. The PCR technique: DNA sequencing. Eaton Publishing Co., Natick, Mass. (This book contains selected articles on sequencing PCR products published in the journal BioTechniques up until March 1992)

• Bej, A. K., M. H. Mahbubani, and R. M. Atlas. 1991. Amplification of nucleic acids by polymerase chain reaction (PCR) and other methods and their applications. Critical Reviews in Biochemistry and Molecular Biology 26:301-334. (Here is an extensive review article with 236 references regarding PCR)

• Linz, U., U. Delling, and H. Rubsamenwaigmann. 1990. Systematic Studies on Parameters Influencing the Performance of the Polymerase Chain Reaction. Journal of Clinical Chemistry and Clinical Biochemistry 28:5-13.

• There is also a Journal entitled "PCR Methods and Applications" published quarterly by Cold Spring Harbor Press. You can subscribe to this journal by writing to:

  Cold Spring Harbor Laboratory Press
  10 Skyline Drive
  Plainview, New York USA 11803-9729

  In the U.S. and Canada, you can call 1-800-843-4388. All other locations can reach them at 516-349-1930 (phone) or 516-349-1946 (FAX).

Here is a list of general pointers as described in Innis1990

1. Try to keep the primer 50% G-C give or take 15%. If overly G-C rich add a string of As or Ts at 5' end; If overly A-T rich, do the same with Gs and Ts.

2. Try to avoid Gs and Cs at 3' end of the primers. This may increase the chance of forming primer dimers.

3. Avoid self-annealing regions within each primer.

4. Compute Tm as sum of 4 C for G/C and 2 for A/T, then subtract 5 C from this value and that is our annealing temp. Naturally, the annealing temp will be that of the primer with the lower value. Differences of 4-6 C do not seem to affect yield of PCR. Ideally you would like the Tm for each primer to match and be within the 70-75 degrees C range.

5. A good practice is to check the target DNA sequence if it is known for mispriming areas. A quick check scanning the sequence of vector for approximately 70% and above homolgy regions can help prevent obtaining multiple contaminating bands in your PCR.

6. Use of a computer program may help eliminate the use of a poorly designed pair of primers.

There are several programs which deal with PCR primer construction, some of which are shareware. Probably the best way to find them is through a search done through a WWW browser. Links from my Homepage to several software locations will allow you to find them easily.

Some of the older programs are:

1. Pgen - for DOS only

PrimerGen only works on IBM-PC(TM), XT, AT, PS/2 and compatibles with EGA or VGA graphics adaptors. It will not work on computers with CGA or Hercules(TM) graphics cards. A hard drive is NOT required and PrimerGen will fit on a 360K 5.25" floppy disk.

PrimerGen contains a sequence editor where amino acid residues are entered. The amino acid sequence must be ONE fragment and cannot be longer than 70 residues. The sequence must be in the ONE LETTER CODE and cannot contain any UNKNOWNS. After the desired amino acid sequence has been entered have the option of saving the sequence to a disk. PrimerGen will also accept and re-edit previously saved sequence files, and also contains a codon preference table editor. You can get this program by anonymous FTP to ftp.bio.indiana.edu. It is found in the molbio/ibmpc directory.

2. Primer - Stanford - Sun Sparcstations only

3. Primer - Whitehead -Unix, Vms (and DOS and Mac if you can compile it)

• IBM PC and compatibles. A PC/AT (286) class machine or better with 640K of RAM.
• Apple Macintosh computers. A Mac II series or SE/30 with a math coprocessor.
• Most Unix workstations including Sun SPARCStation and DEC DECStation systems.
• DEC VAX/VMS computers under VMS Version 5 with VAX C.

• You can get PRIMER from another user, provided you strictly follow the terms
• You can download the most recent version off the Internet via anonymous FTP from genome.wi.edu in the distribution/primer.0.4 directory.
• You can send a request to:

PRIMER c/o The Lander Lab,
Whitehead Institute/MIT
9 Cambridge Center, Cambridge, MA 02142 USA
FAX: (617) 258-6505
E-mail: primer@genome.wi.edu

4. Amplify - MAC only

The author, Bill Engels can be reached at:

Genetics & Medical Genetics
4106 Genetics/Biotechnology Building
University of Wisconson
Phone: (608) 263-2213
lab: (608) 262-5578
FAX: (608) 262-2976
E-mail: wrengels@facstaff.wisc.edu

5. OSP - Unix, X-windows, Vms, DOS, Mac - by snail-mail only.

Here is the abstract from the paper describing OSP, which appeared in Hillier.Green1991, with information on how to obtain the program.

ABSTRACT:

OSP (Oligonucleotide Selection Program) selects oligonucleotide primers for DNA sequencing and the polymerase chain reaction (PCR). The user can specify (or use default) constraints for primer and amplified product lengths, %(G+C), (absolute or relative) melting temperatures, and primer 3' nucleotides. To help minimize non-specific priming and primer secondary structure, OSP screens candidate primer sequences, using user-specifiable cutoffs, against potential base pairing with a variety of sequences present in the reaction, including the primer itself, the other primer (for PCR), the amplified product, and any other sequences desired (e.g., repetitive element sequences in genomic templates, vector sequence in cloned templates, or other primer pair sequences in multiplexed PCR reactions). Base pairing involving the primer 3' end is considered separately from base pairing involving internal sequences. Primers meeting all constraints are ranked by a ``combined score'', a user-definable weighted sum of any of the above parameters. OSP is being routinely and extensively used to select sequencing primers for the C. elegans genome sequencing project, and human genomic PCR primer pairs for the Washington University Genome Center mapping project, with success rates exceeding 96% and 81% respectively. It is available for research purposes from the authors, at no cost, in both text output and interactive graphics (X windows) versions.

AVAILABILITY:

C language source code for OSP is available (for research purposes only) at no cost from the authors, in either the text output version (tested for VAX/VMS, PC, MAC, and SUN Sparcstations), or interactive X windows graphics version (tested for SUN Sparcstations). To obtain OSP please send your postal address either to Phil Green by E-mail (pg@genome.wustl.edu) or (preferably) by FAX to (314) 362-2985 c/o Paula, the secretary handling OSP requests. You must provide a signed licensing agreement (which she will send you) and a stamped addressed mailer with diskette before the program can be sent to you.

"Hot-start" PCR is a method that generally produces cleaner PCR products. Template DNA and primers are mixed together and held at a temperature above the threshold of non-specific binding of primer to template. All the PCR reaction components are added for the extention reaction except one critical reagent (usually the thermostable polymerase).

Just prior to the cycling, the missing component is added to allow the reaction to take place at higher temperature. Due to lack of non-specific hybridization of primers to template, the amplified DNA bands tend to be cleaner; the primers don't have a chance to anneal non-specifically.

This method is difficult to do because the tubes must be kept on a 100 degrees C heat block as your work surface. There are ways to avoid this however. One way is to quickly cool the tubes on ice while adding the component mix. You can then heat the tubes on the pre-warmed thermocycler just before adding the last component. This may not always be successful due to a thermal ramp that may allow non-specific interactions between primer and template.

Hot starts are also done by creating a physical barrier between the essential components, eg. primers and template. This barrier may be created by putting a half-reaction mixture into the bottom of the tube and melting wax over the mix. The wax used can be "PCR Gems" from Perkin-Elmer/Cetus or any number of home-grown waxes (e.g. paraffin or Paraplast). Cooling solidifies the wax, and the missing components can be placed on top. The mixing of the last component then occurs at high temperature only when the wax melts and the top half-mix is added by convection currents within the tubes. The PCR then proceeds as a normal cycle sequence. Another method is to have an antibody attached to the Taq polymerase which inactivates the polymerase until the antibody is destroyed by heat denaturation. See Kellogg1994 and Sharkey1994 for more details about TaqStart antibodies.

Co-solvents have also been used to eliminate artifacts from PCR reactions. For high fidelity, the specificity of primer to template is desirable. Co-solvents such as glycerol, DMSO, and formamide, work to provide highly stringent reactions by changing the Tm of the primer-template hybridization reaction. Co-solvents have various effects on the thermostablility of the polymerase enzyme. Glycerol tends to extend the resistance of Taq enzyme to heat destruction, while formamide lowers enzyme resistance. In some cases, it may be necessary to add single-strand DNA binding protein in order to keep DNA with a high GC content from forming secondary structures. This may also be a problem in cycle sequencing reactions.

The following references give more details about PCR additives: Blanchard1993, Chou1992a, Chou1992b, DAquila1991, Dutton1993, Hengen1996Jantibs, Hengen1997Juntibs, Horton1994, Rapley1992 and Wainwright1993. Also see Question 38 for techniques to amplify high GC-content DNA tmplates.

Arbitrarily Primed PCR (AP-PCR) or Random Amplified Polymorphic DNA (RAPD) are methods of creating genomic fingerprints from species of which little is known about target sequence to be amplified. Strain-specific arrays of DNA fragments (fingerprints) are generated by PCR amplification using arbitrary oligonucleotides to prime DNA synthesis from genomic sites which they fortuitously match or almost match. Generally, two cycles of PCR are performed under conditions of low stringency with a single random orimer, followed by PCR at high stringency with specific primers. DNA amplified is this manner can be used to determine the relatedness of species or for analysis of Restriction Fragment Length Polymorphisms (RFLP). For more information see Welsh.McClelland1990 and Williams1990.

Touchdown PCR involves decreasing the annealling temperature by 1 degree C every second cycle to a 'touchdown' annealing temp which is then used for 10 or so cycles. It was originally intended to bypass more complicated optimization processes for determining optimal annealing temperatures. The idea is that any differences in Tm between correct and incorrect annealing gives a 2-fold difference in product amount per cycle (4-fold per degree C). You therefore enrich for the correct product over any incorrect products.

Another use for this procedure is in determining DNA sequence for a known peptide sequence. The strategy here is to use two sets of degenerate primers that match potential coding sequences at the two ends of a peptide of known sequence. In practice, this requires that you know a stretch of peptide sequence of only 13 amino acids, with left and right primers of 18 nt (6 a.a.) and a space in between of one or more nt. Using these degenerate primers, you do a touchdown PCR. You will get a huge number of products, but you can select the desired product based on size since you know the exact interprimer distance from the peptide sequence. The advantage of this technique is that the touchdown PCR enriches for products containing correct matches between primers and template. If you clone and sequence a dozen PCR products, you can determine the correct coding sequence for the peptide, design an oligo for hybridization, etc. The technique is especially useful for peptide sequences full of ser, lys, and arg (six codons each). For more information see Don1991.

PCR amplification can be can be performed using a phage plaque or bacterial colony picked directly from an agar plate. This is particularly useful for confirmation of mutants after site-directed mutagenesis, sequence tagged site(s) sequence characterization, identification of mutations following random mutagenesis, etc. For more information see Hofmann.Brian1991, Krishnan.Berg1991a, Krishnan.Berg1991b, Krishnan.Berg1993, and Wang1992.

Be careful when selecting the type of toothpicks to be used because some people claim wooden toothpicks can inhibit the PCR. See Lee1995, Hengen1995Aprtibs and Hengen1997Febtibs for more details.

The use of a solid support for attachment of DNA has been used in order to physically separate the single strands of DNA without the use of gel purification. The opposite strands of DNA can then be independently sequenced for verification. More recently, the use of paramagnetic particles has been developed for use as the attachment medium. DNA strands can then be separated by magnetic force and used in conjunction with sequencing techniques. For more information see Fry1992.

Cycle sequencing is a technique that uses a thermal-cycling procedure similar to PCR amplification for obtaining nucleotide sequence information from DNA samples. The advantages of cycle sequencing are that it is unnecessary to clone a particular gene in order to get it's DNA sequence and that it requires very little starting DNA material. The method is very similar to the standard dideoxy sequencing , but uses the elements of PCR for amplifying the terminated oligonucliotides used in the sequencing. For more information see Rao.Saunders1992, Ruano.Kidd1991 and Smith1990.

The vast majority of fluorescent ddNTPs are not incorporated in the PCR products of cycle sequencing . If they are not removed effectively they form an enormous peak at the start of a run and cause streaking artifacts for several hundred bases afterwards, thus seriously degrading the quality of the sequence.

The best way to remove the unincorporated dyes is to use a sephadex G-50 spin column. Make a hole in the bottom of 0.5 ml eppendorf tube using a hot 30 gauge needle and add about 25 ul volume of silanized zirconium glass beads. Pour sephadex G-50 in 0.3 M sodium acetate (5 grams in 60 ml) to the top of the tube. Place the small eppendorf tube inside a larger 1.5 ml eppendorf tube and spin at about 500 rpm for couple of minutes to remove the excess liquid from the matrix. Transfer the smaller tube to a clean siliconized 1.5 ml tube. The DNA sample is added on top of the sephadex matrix and again spun at 1500 RPM for 2 minutes. The eluate is either precipitated by at least 3 volumes of isopropanol or dried in a roto-vac before loading. If you are able to see traces of remaining dye within the cleaned sample, the sequence is usually so poor as to be unusable.

Another method relies on using Sephadex as a separation matrix, but in static columns of pre-determined size. The problem of flow is overcome by having triton X-100 in the mixture which does not affect the sequencing products. The method is described by Rosenthal.Charnock-Jones1992.

This answer comes from Sasha Kraev (bckraev@aeolus.ethz.ch).

This question may be reduced to "How can one prepare a good template from plasmid for polymerase `Sanger sequencing'?". The original paper of Chen.Seeburg1985 described the use of CsCl centrifugation and alkali denaturation that produced DNA ready to be used in the original protocol from Sanger1977 with the Klenow fragment of DNA polymerase I. New DNA polymerases, particularly phage T7 DNA polymerase, has allowed to use miniprep DNA for sequencing in a reliable way.

The most widely used method utilizes a T7 polymerase/Sequenase kit and an alkali denatured miniprep. However, the quality of DNA, produced by any simple miniprep, is subject to variations, the most important of which are particular clone yield, strain used and individual skills.

Since any miniprep procedure results in a defined amount of contaminants in the plasmid DNA, clones with low yield are generally concentrated in a small volume; this increases the chances of elevating the contaminant level to the point where it interferes with the polymerase reaction, particularly with the labelling step. Use of the pre-labelled primer (typically, with T4 polynucleotide kinase and gamma-ATP or with non-radioactive labels ) reportedly gives better results on "simple" minipreps, probably because the labelling step is more sensitive (it is run at low nucleotide concentrations) to contaminants, than the extension-termination step. It is therefore recommended that labelled primers be used with low-yield clones.

A subset of problems with clone yield is the plasmid size. Not only is there a tendency of lower yield with increasing plasmid size, but also it is becoming difficult to dissolve the required molar amount of DNA (typically, 0.5 pmol) in a small initial reaction volume. Viscous DNA solutions are difficult to pipet and mix accurately, which is the second (in addition to low yield) likely reason why this procedure is becoming increasingly error-prone with increasing plasmid size.

The strain dependence of the miniprep quality is usually attiributed to the presence of endA mutation in many newer strains (e.g. XL-1 Blue, DH5alpha, etc.). This eliminates endogenous nuclease contamination in minipreps and is said to improve the quality of sequencing as well. However, those strains usually grow slower because of other associated mutations (e.g. RecA), which may well be another reason why they tend to give better miniprep DNA, since the liquid cultures of these strains just enter the end of the logarithmic phase in an overnight incubation, while older, usually more vigorous, strains are overgrown and partially lysed in an overnight. In fact, the "BIG RED Book" (See Question 5) suggests growing to an optical density of 1.0 at 600 nm. This advice is ingenuous, but it may be quite difficult and inconvenient to follow it in practice, e.g. with many clones at once.

Variations in miniprep quality do exist, and they tend to aggravate the problems described above. A good starting point is to sequence a miniprep of an empty vector, using no less than 3 ug of DNA. If this gives a clear ladder with no grey background and no stops, you may go on with other clones, some of which may not necessarily sequence well under the same conditions.

Two general solutions have been suggested for the problems, described above. First is the cycle sequencing procedure, which requires about 1/20 of the amount of DNA used in a standard protocol (and hence is more forgiving of DNA quality ), and the second, the use of minicolumn chromatography as an aid in "cleaning up" standard minipreps.

Though these columns are generally expensive, they do provide reliable quality of DNA, which is almost completely free of proteins and low molecular weight nucleic acids. However, they do not reliably eliminate chromosomal DNA contamination and do not solve the problems associated with low DNA yield. Again, it is worth while running a control with an empty vector before blaming a column for the bad quality of a partilular clone. Though one should not despise of the really numerous "rapid and simple" protocols that are regularly published in such journals as Nucleic Acid Research, Biotechniques and others, however, none of those has so far provided a one-for-all solution.

Wash your glass plates very well with soap and water, then apply a silanizing agent such as Rain-X or sigmacote (Sigma) with kimwipes to the inside half of one of the glass plates and let air dry. Some people prefer to siliconize both plates, but this generally is not necessary. An alternative to siliconinzing agents is the use of vegetable oil that can be applied by spray. The non-toxic cooking spray PAM can be used once and washed off with detergent. You can also do the same with Pledge furniture polish. To remove the sigmacote or Rain-X, soak the plates in 2M NaOH or with 5% potassium hydroxide in methanol for a few minutes before washing with hot soap and water.

This answer comes from Zophonias O. Jonsson (zjons@vetbio.unizh.ch):

For the care and feeding of sequencing gels...

The question of FIXING:

One of the most frequently asked questions on this newsgroup is "Do I need to fix my sequencing gels?" For those that are in the habit of coating both plates with repel silane (Gel-Slick or Rain-X) the answer is simple. NO! The consensus of the sequencing experts of this group is that this is not only unnecessary, but a waste of time and acetic acid. Concerns were raised that the urea in the gel could cause trouble in humid climates by re-absorbing moisture from the air but, as far as I know, this is not a problem. It has been reported that immersion in water may cause bands to diffuse somewhat, but this is usually not a problem.

A simple protocol for getting your gel off the plates follows: Clean your plates thoroughly and coat with repel silane. After coating wipe the plates 1x with dH2O and 1x with EtOH before casting the gel. After loading and running separate the plates carefully so that the gel sticks to the lower plate. (If it seems to cling tighter to the upper plate when you start pulling them apart, just turn them and start again). If the gel does not come off easily, breaks or folds, it can be immersed in water while the jigsaw/unfolding takes place. If the gel comes off without folding, bathing the gel in water is not necessary. Carefully place a sheet of Whatman 3MM (or some other equally suitable paper) of the correct size on top of the gel. Sandwich it between the glass plates and turn them. If the gel was not immersed in water it usually adheres tightly to the paper, otherwise take care while taking the plates apart again. As soon as you have the gel on the paper, cover it with (Saran) wrap and dry.

You do not have to fix a sequencing gel in order to expose it to X-ray film. Most people simply transfer the gel to a large piece of Whatman[TM] paper after rinsing out the urea in distilled water.

The paper typically used for transfer is Whatman 3MM, however, it is not the only one that works. An alternative to paper is a used piece of X-ray film with the emulsion toward the gel (if the emulsion is only on one side). The gel does not dry as rapidly on a film and it cannot be put in a gel drier since the film is not porous. You can air dry the gel on either paper or used film. If the gel is tacky, as occurs in high humidity areas, use a light dusting of talcum powder to relieve the tackiness.

There are a number of dyes that can replace ethidium bromide, but none that are both non-toxic and as sensitive. Nontoxic alternatives to ethidium bromide for staining DNA in agarose gels are basic dyes such as methylene blue, toluidine blue, azure A, and brilliant cresyl blue described by Santillan-Torres1993. For a discussion on current practices for disposal of ethidium bromide, see Hengen1994Juntibs.

A more sensitive stain is SYBR Green I from Molecular Probes. This dye is extremely sensitive, but costs much more than ethidium bromide. Two forms of SYBR Green are availible. SYBR Green I is recommended for DNA and SYBR Green II is recommended for single-stranded nucleic acids (including RNA). Both can be used with a standard UV transilluminator (300 nm) but are much more sensitive when used with a 254 nm illuminator (typically an epi-illuminator).

SYBR Green I from Molecular Probes can be used with either agarose or polyacrylamide gels, and is particularly suitable for low amount of small < 100 bp PCR fragments as these are difficult to visualize with ethidium bromide. Any DNA fragment that can be seen with ethidium bromide can be seen with SYBR Green I, so for normal use it's not worth the added expense. SYBR Green I (Catalogue Number S-7567, 1 ml in DMSO) goes for $195 and SYBR Green II (Catalogue Number S-7568, 1 ml in DMSO) for $195. The manufacturer recommends a 1:5000 or 1:10000 dilution for staining. In addition, you will need to purchase a green photographic filter (Catalogue Number S-7569, $29) to photograph the gels. Alternatively, a Wratten Gelatin filter No. 15 (Kodak Catalog number 149 5548) can be used. It goes for about $6.95 US.

There are some shortcomings compared with using ethidium bromide, however. First, if you stain your gel with SYBR Green I, it is nearly impossible to remove the dye because you can't extract it with an organic solvent like isopropanol or isoamyl alcohol. For example, if you wish to extract DNA from a gel for a mobility shift, ethidium bromide is probably better since it can be removed and shouldn't interfere with DNA binding proteins. Second, it is very expensive and has a very short lifespand. Over a short time (less than one year in DMSO at -20 C) it begins to lose it's effective staining ability. Even when aliquoted it into small volumes of 20 ul and used one at a time, the sensitivity drops over time. Adding more dye usually fixes the problem in the short term, but you will most likely have to buy a fresh batch of dye. On the better end, when fresh it is much more sensitive than EtBr and is less mutagenic according to the Ames test for auxotrophic reversion frequencies. In the Ames test, SYBR Green I produced an approximately 2-fold increase in histidine (+) revertants in S. typhimurium strains TA98 and TA102. No mutagenic activity was observed in strains TA100, TA1535, TA1537, TA1538, and TA97a. EtBr produced an approximate increase of 70-fold in strain TA98, 15-fold in TA1537, 4-fold in TA97a, 80-fold in TA 1538, and 2-fold in TA102. No activity was observed in TA100 or TA1535. These results have not yet been published.

For information about all kinds of dyes, get a catalog from Molecular Probes and be impressed! Also, FMC BioProducts now sells SYBR Green I and SYBR Green II under agreement with Molecular Probes.

Another alternative is Acridine Orange. This substance can bind to double-stranded nucleic acid by intercalation or can bind single- and double-stranded nucleic acid by electrostatic interaction with the phosphate.

Ultraviolet irradiation adsorbed at 260 nm can be fluoresced by double-stranded nucleic acid at 530 nm (green) or by single-stranded nucleic acid at 640 nm (red), which allows one to distinguishing the two. Staining of gels can be done with 30 micrograms/ml of Acridine Orange for 1 hour in 10 mM salt and destaining for 1 hour in 0.1 mM salt. However, one problem is that Acridine Orange is as mutagenic as ethidium bromide by the Ames test. For more information about the use of Acridine Orange, see McMaster1977, Carmichael1980 and Ogden1987.

This answer comes from Song Tan (tan@mol.biol.ethz.ch):

A good reference for determining protein concentration from UV absorption measurements is Gill.von.Hippel1989. These workers assume the proteins are fully denatured in 6 M GuHCl and calculate extinction coefficients of proteins based on their tryptophan, tyrosine and cysteine content. They also show that the difference between the calculated extinction coefficient for denatured protein and the measured extinction coefficient for native protein is very small (confirmed for a wide selection of proteins). If you have access to GCG, the command PEPTIDESORT will calculate the molar extinction coefficent based on the Gill and von Hippel formula.

To get to an alphabetical listing of Material Safety Data Sheets, you can use gopher by typing the following:

gopher atlas.chem.utah.edu

gopher ginfo.cs.fit.edu:70/1m/safety/msds

• http://physchem.ox.ac.uk/MSDS/
  
• http://www.fisher1.com/Fisher/Alphabetical_Index.html
  
• http://www.pp.orst.edu
  
• http://unixg.ubc.ca:780/~bchydro/bchydro.html
  
• http://cansnd.cisti.nrc.ca/~nash/msds.html
  

Some people feel the need to break up the methods newsgroup because the traffic has increased dramatically over the past few years. However, this is mostly a problem for people who get the posted articles by E-mail and not through a threaded newsreader. The first suggestion if you are having overload problems is to find your system administrator and have him/her set you up correctly with newsreader software.

There are definite advantages to not breaking up bionet.molbio.methds-reagnts.

1. Most methods and techniques discussed are ubiquitous to all molecular biology. People with diverse backgrounds are all meeting in one place and this gives a great spread of interests and approaches to solving problems. If broken up, we lose major contributors and therefore their insights.

2. With many different groups that discuss methods, people begin to cross-post messages in order to reach a wider audience. Cross-posting then becomes so rampant that you might as well be subscribed to only one newsgroup anyway. If you are subscribed to a few different groups, you end up re-reading most of the postings. This becomes tediously boring and wastes a huge amount of time and effort.

The first step in locating the sequence for any given plasmid is to check GenBank or EMBL sequence database for it! This can be done through gopher or by using a WWW client pointed to http://www.ncbi.nlm.nih.gov. If the plasmid is sold by a Biotech company, the sequence might be made available at an FTP site for that company. Try using Martin Leach's BioTechnology Company Listing at http://www.data-transport.com, then select biotech registry, or go through Paul Hengen's Homepage at http://www-lmmb.ncifcrf.gov/~pnh/ to Martin's site in order to locate the company's archive site.

Another useful site chock full of vector sequences is maintained by Stephen Misener at Queens University at Kingston located at http://biology.queensu.ca/~miseners/vector.html.

One option is to contact the author of the paper where the vector was originally described. That person might be holding the entire DNA sequence. Because it takes extra effort to submit a sequence to the database and no one seems to get any kind of credit for it, it is likely that many people will continue to hold on to sequences and distribute them personally either on floppy diskette or by E-mail. Having exhausted all other possiblilites, someone in the methods newsgroup might be able to provide a copy of the sequence to you, but beware that many poorly copied or hand-created sequences with many errors are being propagated this way. This happens frequently and you should definitely be aware of this. You've been warned!

Poor or slow polymerization of acrylamide is most likely the result of an older solution of ammonium persulfate (APS) solution which has gone off. First try making a fresh stock from solid material. Netters have several quick tests to see if the APS is still good. One way is to put some in a test tube and add the correct volume of water to make 10 mg/ml. If you hear it snap, crackle, and pop, then it is still good. This is because some gas forms within the crystals when water is added to APS which causes them to pop. Another way is to prepare solution of NaI in water and add your APS to it. If the color changes to brown, the APS is still good. Opinions vary concerning how long APS is stable. Some netters store a 10% stock of APS at 4 degrees C for several weeks, while others make 10% APS and freeze single use aliquots at -20. Still others make just enough fresh solution for a single gel just before pouring one. If all tests for polymerization fail, the next thing to test is the solution of TEMED. Buy a fresh bottle and start over.

Satellite or "feeder" colonies appear on ampicillin plates near ampicillin resistant colonies because the enzyme responsible for antibiotic resistance, beta-lactamase, is secreted from the cells. The use of other penicillin derivatives containing beta-lactam rings sometimes alleviates this. It is a matter of debate whether the enzyme is inhibited from leaving the cell, or the derivatives are more resistant to the secreted form of the enzyme. In any case, the number of feeder colonies can be reduced by using 200 ug/ml carbenicillin, or a mixture of ampicillin 20 ug/ml and methicillin 80 ug/ml.

Here is a list of suppliers that sell carbenicillin from the the 1996 Cold Spring Harbor Lab Manual Source Book. The Source Book is a buyer's guide for lab reagents, supplies and equipment. It is distributed free from CSHL Press. You can request a free copy at the Source Book's WWW site http://www.biosupplynet.com.

• Accurate Chemical & Scientific Corp 800-645-6264
  
• American Bioanalytical Inc. 800-4443-0600
  
• BioWorld 800-860-9729
  
• Crescent Chemical Co. 800-645-3412
  
• Life Technologies Inc. (GIBCO BRL) 800-828-6686
  
• Sigma Chemical Co. 800-325-3010
  
• USB Specialty Biochemicals 800-321-9322
  
• Wako Chemicals USA Inc. 800-992-9256
  

One problem with cycle sequencing is that GC-rich sequences begin forming secondary structures which cause polymerase pausing and stoppage, resulting in bands in all four lanes (BAFLs). The problem seems to be more troublesome when using commercial Taq buffers that contain potassium. See Hengen1996Jantibs and Woodford1995 for more information. Also see Question 21 for references concerning additives to the PCR.

The following list was collected by me for people who would like to contact technical service reps by E-mail. An additional list of biotechnology companies available on the World Wide Web from the
BioTechnology Company Registry.

Paul N. Hengen's List of Biotechnology Service Representatives:

Aldrich Chemical Company
1001 West St. Paul Ave.
Milwaukee, WI 53233 USA
Phone: (414) 273-3850 ext 5700 tech service
Phone: (800) 231-8327 direct to tech service
FAX: (414) 273-4979 ATTN: tech service
FAX: (800) 962-9591 ATTN: tech service

Ambion, Inc.
E-mail: techserv@ambion.com

Amersham Life Science - USB
Barbara Grossmann
Phone: (800) 321-9322 x142
FAX: (216) 360-0974
E-mail: dr277@cleveland.freenet.edu

Amersham Corporation
Will Volny
Phone: 1 (800) 341-7543
FAX: 1 (708) 437-1640
E-mail: p00475@psilink.com

Applied Biosystems
Morgan Conrad
Phone: (415) 570-6667
E-mail: mpc@apldbio.com
E-mail: info@apldbio.com for technical service
E-mail: biobytes@apldbio.com for computational information

Biometra, Inc.
Tony Sanchez
Phone: (800) 932-7250
FAX: (813) 287-5163
E-mail: biometra@gate.net

Bio-Rad Laboratories
Connie Rickey
Phone: (510) 741-6781
E-mail: crickey@haley.genetics.bio-rad.com

Biozym Nederland bv
P.O. Box 31087
6370 AB Landgraaf
The Netherlands
Leon de Bruin
Phone: +31+(0)45-5327755
FAX: +31+(0)45-5327733
E-mail: biozymbv@cuci.nl

Clontech Laboratories, Inc.
4030 Fabian Way
Palo Alto
CA 94303-9605, U.S.A.
E-mail: tech@clontech.com

Epicentre Technologies Corp.
Madison, WI
Julie Kramer (Technical Services Manager)
Ronald Meis (Applications Scientist)
Phone: (800) 284-8474
FAX: (608) 258-3088
E-mail: techhelp@epicentre.com

Ericomp, Inc.
Jim Whitney
Phone: (800) 541-8471 (in the U.S.)
Phone: (619) 457-1888 (outside the U.S.)
FAX: (619) 457-2937
E-Mail: ericompladel@delphi.com
Web Page: http://www.ericomp.com

Fluka Chemical Corporation
980 South Second Street
Ronkonkoma, NY 11779-7238 USA
Phone: (516) 467-0980 ext 4163 tech service
Phone: (800) 358-5287 ext 4163 tech service
FAX: (516) 467-0663 ATTN: tech service
FAX: (800) 441-8841 ATTN: tech service

FMC BioProducts
191 Thomaston St.
Rockland, ME 04841
Doug Robinson
Phone: (800) 521-0390
FAX: (800) 362-1133
Customer Services
Phone: (800) 341-1574
FAX: (800) 362-5552
E-mail: biotechserv@fmc.com

GenHunter Corporation
624 Grassmere Park Drive, Suite 17
Nashville, TN 37211
phone: (615) 833-0665
FAX: (615) 832-9461

Genomed, Inc.
Rusty Soots
Phone: (800) 436-6548
FAX: (919) 870-9352
E-mail: glennsey@rock.concert.net

GenPharm
Tony Cruz
Phone: (415) 964-7024
FAX: (415) 964-3537
E-mail: tcruz@genpharm.com

GraphPad Software
Paige Searle & Dr. Harvey Motulsky
Phone:(800) 388-GPAD or 619-457-3909
FAX: 619-457-8141
E-mail: sales@graphpad.com
E-mail: support@graphpad.com
HomePage: http://www.graphpad.com

Life Science Resources
Rusty Soots
Phone: (919) 481-4718
FAX: (919) 870-9352
E-mail: glennsey@rock.concert.net

Life Technologies, Inc. (GIBCO-BRL)
[Formarly Bethesda Research Labs (BRL)]
Joseph Crouse
Phone: (301) 840-4135
FAX: (301) 670-8599
E-mail: GIBCO BRL@aol.com

MJ Research
Michael Finney
Phone: (800) 729-2165
FAX: (617) 923-8080
Outside USA, call local distributor.
E-mail: tech@mjr.com
E-mail: sales@mjr.com

Molecular Probes, Inc.
P.O. Box 22010
4480 Pitchford Ave.
Eugene OR 97402 USA
Phone: (503) 465-8338
Phone: (503) 465-8300
FAX: (503) 344-6504
Phone: (800) 438-2209 (Orders)
FAX: (800) 438-0228 (Canada & U.S. only) Technical Assistance
Phone: (503) 465-8353
FAX: (503) 344-6504
E-mail: tech@probes.mhs.compuserve.com

Molecular Probes Europe BV 4 in Europe:
PoortGebouw, Rijnsburgerweg 10
2333 AA Leiden, The Netherlands
FAX: +31 71 233419
Toll free FAX number for orders (from UK): 00 31 800 5551

The Nest Group
Value Added Resellers for Macherey-Nagel, manufacturer of Nucleobond AX
Amos Heckendor
phone: (800) 347-6378
phone: (508) 481-6223
E-mail: nestgrp@world.std.com

New England BioLabs, Inc. (NEB - U.S. headquarters)
Telephone orders: 1-800-632-5227 (1-800-NEB-LABS)
Technical assistance: 1-800-632-7799
FAX: 1-508-921-1350 (orders and technical assistance)
E-mail: info@neb.com

NEB Subsidiaries:
New England BioLabs GmbH, Germany
Tel. (0130) 83 30 31
E-mail: info@de.neb.com

New England BioLabs Ltd., Canada
Tel. (800) 387-1095
E-mail: info@ca.neb.com

New England BioLabs (UK) Ltd.
Tel. (0800) 31 84 86
E-mail: info@uk.neb.com

Novagen, Inc.
597 Science Drive
Madison, WI 53711
608-238-6110
Order/Proc. 800-526-7319
Tech support: 800-207-0144
FAX tech support: 608-232-2288
Email tech support: Novatech@Novagen.com
World-wide Web Home Page: http://www.novagen.com

PanVera Corporation
565 Science Drive
Madison, WI 53711
Toll-free Ordering & Technical Service: 800-791-1400
Ordering & Technical Service: 608-233-5050
FAX: 608-233-3007
E-mail: info@panvera.com
HomePage: http://www.panvera.com/

Promega Corporation
2800 Woods Hollow Road
Madison, Wisconsin 53711-5399
Telephone in US: 800-356-9526
FAX in US: 800-926-1166
Telephone outside US: 608-274-4330
FAX outside US: 608-273-6967
E-Mail: techserv@promega.com
HomePage: http://www.promega.com/

QIAGEN, Inc.
Kirk Malloy, PhD.
Phone: (800) 362-7737
FAX: (805) 295-7654
E-mail: techservice-us@qiagen.de
HomePage: http://www.qiagen.com/

Schleicher & Schuell (S+S)
Janet LaRoche
Phone: (603) 352-3810
FAX: (603) 357-3627
Telephone orders & Technical assistance: 1-800-245-4024
FAX: (orders and technical assistance): 1-603-357-7700
E-mail: kieron@sands.mv.com

Sigma Chemical Company
3050 Spruce Street
St. Louis, MO 63103 USA
Phone: (314) 771-5765 ext 3901 rsch tech service
Phone: (800) 325-5832 direct to rsch tech service
FAX: (314) 771-3814 direct to rsch tech service
FAX: (800) 325-5052 ATTN: research tech service
E-mail: sigma-techserv@sial.com

Sigma Diagnostics
545 South Ewing
St. Louis, MO 63103 USA
Phone: (314) 771-5765 ext 2950 clin tech service
Phone: (800) 325-0250 direct to clin tech service
FAX: (314) 652-9930 direct to clin tech service
E-mail: sigma-clintech@sial.com

Stratagene
Sam Marsh
Phone: (800) 424-5444 x4400
FAX: (619) 535-0034
E-mail: sam_marsh@stratagene.com
E-mail: tech_services@stratagene.com

Supelco, Inc.
Supelco Park
Belleftone, PA 16823-0048 USA
Phone: (814) 359-3041 direct to tech service
Phone: (800) 359-3041 direct to tech service
FAX: (814) 359-5468 direct to tech service
FAX: (800) 447-3044 ATTN: tech service

Synapsys Corp.
FAX: (617) 272-6179
E-mail: synapsys@world.std.com

If anyone else would like to be added to the list, please contact pnh@ncifcrf.gov.

---------- List of References Cited in BiBTeX format ----------

@article{Birnboim1979,
author = "H. C. Birnboim
and J. Doly",
title = "A rapid alkaline extraction procedure for screening
recombinant plasmid {DNA}",
journal = "Nucl. Acids Res.",
volume = "7",
pages = "1513-1523",
year = "1979"}

@article{Birnboim1983,
author = "H. C. Birnboim",
title = "A rapid alkaline extraction method for the
isolation of plasmid {DNA}",
journal = "Meth. Enzym.",
volume = "100",
pages = "243-255",
year = "1983"}

@article{Blanchard1993,
author = "M. M. Blanchard
and P. Taillon-Miller
and P. Nowotny
and V. Nowotny",
title = "{PCR} buffer optimization with uniform
temperature regimen to facilitate automation",
journal = "PCR Methods and Applications",
volume = "2",
pages = "234-240",
year = "1993"}

@article{Bolivar1988,
author = "F. Bolivar",
title = "Plasmid {pBR322}: the multipurpose cloning vector",
journal = "B.R.L. Focus",
volume = "10",
pages = "61-64",
year = "1988"}

@article{Carmichael1980,
author = "G. C. Carmichael
and G. K. McMaster",
title = "The analysis of nucleic acids in gels using
glyoxal and acridine orange",
journal = "Meth. Enzymol.",
volume = "65",
pages = "380-391",
year = "1980"}

@article{Carter1993,
author = "M. J. Carter
and I. D. Milton",
title = "An inexpensive and simple method for {DNA}
purifications on silica particles",
journal = "Nucleic Acids Res.",
volume = "21",
number = "4",
pages = "1044",
comment = "diatomaceous earth used as matrix for Gene Clean",
year = "1993"}

@article{Chen.Seeburg1985,
author = "E. Y. Chen
and P. H. Seeburg",
title = "Supercoil sequencing: a fast and simple
method for sequencing plasmid {DNA}",
journal = "DNA",
volume = "4",
pages = "165-170",
year = "1985"}

@article{Chou1992b,
author = "Q. Chou",
title = "Minimizing deletion mutagenesis artifact during
{{\em Taq}} {DNA} polymerase {PCR} by {{\em E. coli}} {SSB}",
journal = "Nucleic Acids Res.",
volume = "20",
number = "16",
pages = "4371",
comment = "cycle sequencing through high GC content",
year = "1992"}

@article{Chuang1995,
author = "S.-E. Chuang
and A.-L. Chen
and C.-C. Chao",
title = "Growth of {{\em E. coli}} at low temperature
dramatically increases the transformation frequency by
electroporation",
journal = "Nucleic Acids Res.",
volume = "23",
number = "9",
pages = "1641",
year = "1995"}

@article{Chung1989,
author = "C. T. Chung
and S. L. Niemela
and R. H. Miller",
title = "One--step preparation of competent {{\em Escherichia coli}}:
transformation and storage of bacterial cells in the same solution",
journal = "Proc. Natl. Acad. Sci. USA",
volume = "86",
pages = "2172-2175",
month = "April",
comment = "also see Epicentre Technologies Forum vol.2, no.3, pg.5",
year = "1989"}

@article{Cohen1972,
author = "S. N. Cohen
and A. C. Y. Chang
and L. Hsu",
title = "Nonchromosomal antibiotic resistance in bacteria:
genetic transformation of {{\em Escherichia coli}} by {R-factor} {DNA}",
journal = "Proc. Natl. Acad. Sci. USA",
volume = "69",
pages = "2110-2114",
year = "1972"}

@article{DAquila1991,
author = "R. T. D'Aquila
and L. J. Bechtel
and J. A. Videler
and J. J. Eron
and P. Gorczyca
and J. C. Kaplan",
title = "Maximizing sensitivity and specificity
of {PCR} by preamplification heating",
journal = "Nucl. Acids Res.",
volume = "19",
number = "13",
pages = "3749",
comment = "hot start PCR",
year = "1991"}

@article{Chou1992a,
author = "Q. Chou
and M. Russell
and D. E. Birch
and J. Raymond
and W. Bloch",
title = "Prevention of pre-{PCR} mis-priming and primer
dimerization improves low-copy-number amplifications",
journal = "Nucl. Acids Res.",
volume = "20",
number = "7",
pages = "1717-1723",
comment = "hot start PCR",
year = "1992"}

@article{Dagert1979,
author = "M. Dagert
and S. D. Ehrlich",
title = "Prolonged incubation in calcium chloride
improves the competence of {{\em Escherichia coli}} cells",
journal = "Gene",
volume = "6",
pages = "23-28",
year = "1979"}

@article{Don1991,
author = "R. H. Don
and P. T. Cox
and B. J. Wainwright
and K. Baker
and J. S. Mattick",
title = "Touchdown {PCR} to circumvent spurious priming
during gene amplification",
journal = "Nucl. Acids Res.",
volume = "19",
pages = "4008",
year = "1991"}

@article{Dower1988,
author = "W. J. Dower
and J. F. Miller
and C. W. Ragsdale",
title = "High efficiency transformation of {{\em E. coli}}
by high voltage electroporation",
journal = "Nucleic Acids Res.",
volume = "16",
number = "13",
pages = "6127-6145",
year = "1988"}

@article{Dutton1993,
author = "C. M. Dutton
and C. Paynton
and S. S. Sommer",
title = "General method for amplifying regions of very
high {G + C} content",
journal = "Nucleic Acids Res.",
volume = "21",
number = "12",
pages = "2953-2954",
comment = "cycle sequencing through high GC content",
year = "1995"}

@article{Eynard1992,
author = "N. Eynard
and S. Sixou
and N. Duran
and J. Teissie",
title = "Fast kinetics studies of {{\em Escherichia coli}}
electrotransformation",
journal = "Eur. J. Biochem",
volume = "209",
pages = "431-436",
year = "1992"}

@article{Fry1992,
author = "G. Fry
and E. Lachenmeier
and E. Mayrand
and B. Giusti
and J. Fisher
and L. Johnston-Dow
and R. Cathcart
and E. Finne
and L. Kilaas",
title = "A new approach to template purification
for sequencing applications using paramagnetic particles",
journal = "BioTechniques",
volume = "13",
pages = "124-131",
year = "1992"}

@article{Gill.von.Hippel1989,
author = "S. C. Gill
and P. H. {von Hippel}",
title = "Calculation of protein extinction coefficients
from amino acid sequence data",
journal = "Anal Biochem",
volume = "182",
pages = "319-326",
year = "1989"}

@article{Hanahan1983,
author = "D. Hanahan",
title = "Studies in transformation of
{{\em Escherichia coli}} with plasmids",
journal = "J. Mol. Biol.",
volume = "166",
pages = "557-580",
comment = "Landmark paper on chemical transformation of bacteria",
year = "1983"}

@inbook{Hanahan1985,
author = "D. Hanahan",
editor = "D. M. Glover",
title = "{DNA} cloning: A practical approach",
chapter = "Techniques for transformation of {{\em E. coli}}",
publisher = "IRL Press",
volume = "I",
address = "Oxford",
pages = "109-135",
year = "1985"}

@article{Hengen1994Juntibs,
author = "P. N. Hengen",
title = "Methods and reagents - Disposal of ethidium bromide",
journal = "Trends in Biochemical Sciences",
volume = "19",
number = "6",
pages = "257-258",
month = "June",
year = "1994"}

@article{Hengen1994Octtibs,
author = "P. N. Hengen",
title = "Methods and reagents - Better competent cells and
{DNA} polymerase contaminants",
journal = "Trends in Biochemical Sciences",
volume = "19",
number = "10",
pages = "426-427",
month = "October",
year = "1994"}

@article{Hengen1994Septibs,
author = "P. N. Hengen",
title = "Methods and reagents - Recovering {DNA} from agarose gels",
journal = "Trends in Biochemical Sciences",
volume = "19",
number = "9",
pages = "388-389",
month = "September",
year = "1994"}

@inproceedings{Hengen1995,
author = "P. N. Hengen",
editor = "A. M. Griffin
and H. G. Griffin",
booktitle = "Molecular Biology: Current Innovations and Future Trends",
title = "Mini-prep plasmid {DNA} isolation and purification
using silica-based resins",
publisher = "Horizon Scientific Press",
volume = "Part 1",
address = "Wymondham, U.K.",
pages = "39-50",
year = "1995"}

@article{Hengen1995Aprtibs,
author = "P. N. Hengen",
title = "Methods and reagents - Caring for your hybridization membranes",
journal = "Trends in Biochemical Sciences",
volume = "20",
number = "4",
pages = "160-161",
month = "April",
year = "1995"}

@article{Hengen1995Juntibs,
author = "P. N. Hengen",
title = "Methods and reagents - Electro-transformation of
{{\em Escherichia coli}} with plasmid {DNA}",
journal = "Trends in Biochemical Sciences",
volume = "20",
number = "6",
pages = "248-249",
month = "June",
year = "1995"}

@article{Hengen1996Febtibs,
author = "P. N. Hengen",
title = "Methods and reagents - Preparing ultra--competent
{{\em Escherichia coli}}",
journal = "Trends in Biochemical Sciences",
volume = "21",
number = "2",
pages = "75-76",
month = "February",
year = "1996"}

@article{Hengen1996Jantibs,
author = "P. N. Hengen",
title = "Methods and reagents - Cycle sequencing through
{GC}--rich regions",
journal = "Trends in Biochemical Sciences",
volume = "21",
number = "1",
pages = "33-34",
month = "January",
year = "1996"}

@article{Hengen1997Febtibs,
author = "P. N. Hengen",
title = "Methods and reagents - Emergency sterilization using
microwaves",
journal = "Trends in Biochemical Sciences",
volume = "22",
number = "2",
pages = "68-69",
month = "February",
year = "1997"}

@article{Hengen1997Juntibs,
author = "P. N. Hengen",
title = "Methods and reagents - Optimizing multiplex and {LA-PCR}
with betaine",
journal = "Trends in Biochemical Sciences",
volume = "22",
number = "6",
pages = "225-226",
month = "June",
year = "1997"}

@article{Heyd1996,
author = "M. L. Heyd
and J. D. Diehl Jr.",
title = "Recovering {DNA} from agarose gels with pumice",
journal = "BioTechniques",
volume = "20",
number = "3",
pages = "394-398",
year = "1996"}

@article{Hillier.Green1991,
author = "L. Hillier
and P. Green",
title = "{OSP}: A Computer Program for Choosing {PCR}
and {DNA} Sequencing Primers",
journal = "PCR Methods and Applications",
volume = "1",
number = "2",
pages = "124-128",
year = "1991"}

@article{Hofmann.Brian1991,
author = "M. A. Hofmann
and D. A. Brian",
title = "Sequencing {PCR} {DNA} amplified directly
from a bacterial colony",
journal = "BioTechniques",
volume = "11",
pages = "30-31",
year = "1991"}

@article{Horton1994,
author = "R. M. Horton
and B. L. Hoppe
and B. M. Conti-Tronconi",
title = "Ampli{G}rease: ``hot start'' {PCR} using
petroleum jelly",
journal = "BioTechniques",
volume = "16",
number = "1",
pages = "42-43",
comment = "hot start PCR",
year = "1994"}

@inbook{Innis1990,
author = "M. A. Innis
and D. H. Gelfand",
editor = "M. A. Innis
and D. H. Gelfand
and J. J. Sninsky
and T. J. White",
title = "{PCR} Protocols: A Guide to Methods and Applications",
chapter = "Optimization of PCRs",
publisher = "Academic Press",
address = "San Diego",
pages = "3-12",
year = "1990"}

@article{Inoue1990,
author = "H. Inoue
and H. Nojima
and H. Okayama",
title = "High efficiency transformation of
{{\em Escherichia coli}} with plasmids",
journal = "Gene",
volume = "96",
pages = "23-28",
year = "1990"}

@article{Jupin1995,
author = "I. Jupin
and B. Gronenborn",
title = "Abundant, easy and reproducible production of
single-stranded {DNA} from phagemids using helper
phage-infected competent cells",
journal = "Nucleic Acids Res.",
volume = "23",
number = "3",
pages = "535-536",
comment = "transformation of cells with phagemid DNA",
year = "1995"}

@article{Kellogg1994,
author = "D. E. Kellogg
and I. Rybalkin
and S. Chen
and N. Mukhamedova
and T. Vlasik
and P. D. Siebert",
title = "{TaqStart Antibody}^{\scriptsize {TM}}: {''Hot Start''}
{PCR} facilitated by a neutralizing monoclonal antibody directed
against {{\em Taq}} {DNA} polymerase",
journal = "BioTechniques",
volume = "16",
number = "6",
pages = "1134-1137",
month = "June",
comment = "antibodies against Taq polymerase; Hot start",
year = "1994"}

@article{Krishnan.Berg1991a,
author = "B. R. Krishnan
and R. W. Blakesley
and D. E. Berg",
title = "Linear amplification {DNA} sequencing directly
from single phage plaques and bacterial colonies",
journal = "Nucleic Acids Res.",
volume = "19",
number = "5",
pages = "1153",
year = "1991"}

@article{Krishnan.Berg1991b,
author = "B. R. Krishnan
and D. Kersulyte
and I. Brikun
and C. M. Berg
and D. E. Berg",
title = "Direct and crossover {PCR} amplification to
facilitate Tn{/em 5supF}-based sequencing of lambda phage clones",
journal = "Nucleic Acids Res.",
volume = "19",
number = "22",
pages = "6177-6182",
year = "1991"}

@article{Krishnan.Berg1993,
author = "B. R. Krishnan
and D. Kersulyte
and I. Brikun
and H. V. Huang
and C. M. Berg
and D. E. Berg",
title = "Transposon-based and polymerase chain
reaction-based sequencing of {DNAs} cloned in lambda phage",
journal = "Meth. Enzym.",
volume = "218",
pages = "258-279",
year = "1993"}

@article{Kubiniec1990,
author = "R. T. Kubiniec
and H. Liang
and S. W. Hui",
title = "Effects of pulse length and pulse strength
on transfection by electroporation",
journal = "BioTechniques",
volume = "8",
number = "1",
pages = "16-20",
year = "1990"}

@article{Lee1995,
author = "A. B. Lee
and T. A. Cooper",
title = "Improved direct {PCR} screen for bacterial
colonies: wooden toothpicks inhibit {PCR} amplification",
journal = "BioTechniques",
volume = "18",
number = "2",
pages = "225-226",
year = "1995"}

@article{Liu1990,
author = "H. Liu
and A. Rashidbaigi",
title = "Comparison of various competent cell preparation
methods for high efficiency {DNA} transformation",
journal = "BioTechniques",
volume = "8",
pages = "21-25",
year = "1990"}

@book{Maniatis1982,
author = "T. Maniatis
and E. F. Fritsch
and J. Sambrook",
title = "Molecular Cloning, A Laboratory Manual",
publisher = "Cold Spring Harbor Laboratory",
address = "Cold Spring Harbor, New York",
year = "1982"}

@article{McMaster1977,
author = "G. K. McMaster
and G. C. Carmichael",
title = "Analysis of single- and double- stranded nucleic acids
on polyacrylamide and agarose gels by using glyoxal and Acridine Orange",
journal = "Proc. Natl. Acad. Sci. USA",
volume = "74",
pages = "4835-4838",
year = "1977"}

@article{Messing1982,
author = " J. Messing
and J. Vieira",
title = "A new pair of {M13} vectors for selecting either {DNA}
strand of double-digest restriction fragments",
journal = "Gene",
volume = "19",
pages = "269-276",
year = "1982"}

@article{Morelle1989,
author = "G. Morelle",
title = "A plasmid extraction procedure on a miniprep scale",
journal = "B.R.L. Focus",
volume = "11",
pages = "7-8",
year = "1989"}

@article{Nelson1989,
author = "M. Nelson
and M. McClelland",
title = "Effect of site-specific methylation on {DNA}
modification methyltransferases and restriction endonucleases",
journal = "Nucleic Acids Res.",
volume = "17",
pages = "389-415",
year = "1989"}

@article{Nishimura1990,
author = "A. Nishimura
and M. Morita
and Y. Nishimura
and Y. Sugino",
title = "A rapid and highly efficient method for preparation of
competent {{\em Escherichia coli}} cells",
journal = "Nucleic Acids Res.",
volume = "18",
pages = "6169",
year = "1990"}

@article{O'Callaghan1990,
author = "D. O'Callaghan
and A. Charbit",
title = "High efficiency transformation of
{{\em Salmonella typhimurium}} and {{\em Salmonella typhi}}
by electroporation",
journal = "Mol. Gen. Genet.",
volume = "223",
pages = "156-158",
year = "1990"}

@article{Ogden1987,
author = "R. C. Ogden
and D. A. Adams",
title = "Electrophoresis in agarose and acrylamide gels",
journal = "Meth. Enzymol.",
volume = "152",
pages = "61-87",
comment = "acridine orange",
year = "1987"}

@article{Okayama1987,
author = "H. Okayama
and M. Kawaichi
and M. Brownstein
and F. Lee
and T. Yokota
and K. Arai",
title = "High-efficiency cloning of full-length {cDNA}; construction
and screening of {cDNA} expression libraries for mammalian cells",
journal = "Meth. Enzym.",
volume = "154",
pages = "3-28",
year = "1987"}

@article{Parker1977,
author = "R. C. Parker
and R. M. Watson
and J. Vinograd",
title = "Mapping of closed circular {DNAs} by cleavage with restriction
endonucleases and calibration by agarose gel electrophoresis",
journal = "Proc. Natl. Acad. Sci. USA",
volume = "74",
pages = "851-855",
comment = "partial digests using ethidium bromide",
year = "1977"}

@article{Parker1980,
author = "R. C. Parker",
title = "Conversion of circular {DNA} to linear strands for mapping", journal = "Meth. Enzym.",
volume = "65",
pages = "415-426",
year = "1980"}

@article{Nelson1991,
author = "M. Nelson
and M. McClelland",
title = "Site-specific methylation: effect on {DNA} modification
methyltransferases and restriction endonucleases",
journal = "Nucleic Acids Res.",
volume = "19",
pages = "2045-2071",
year = "1991"}

@article{Potter1993,
author = "H. Potter",
title = "Application of electroporation in recombinant {DNA}
technology",
journal = "Meth. Enzymol.",
volume = "217",
pages = "461-478",
year = "1993"}

@article{Rao.Saunders1992,
author = "V. B. Rao
and N. B. Saunders",
title = "A rapid polymerase-chain-reaction-directed
sequencing strategy using a thermostable {DNA} polymerase
from {{\em Thermus flavus}}",
journal = "Gene",
volume = "113",
pages = "17-23",
year = "1992"}

@article{Ruano.Kidd1991,
author = "G. Ruano
and K. K. Kidd",
title = "Coupled amplification and sequencing of
genomic {DNA}",
journal = "Proc. Natl. Acad. Sci. U.S.A.",
volume = "88",
pages = "2815-2819",
year = "1991"}

@article{Smith1990,
author = "D. P. Smith
and E. M. Johnstone
and S. P. Little
and H. M. Hsiung",
title = "Direct {DNA} sequencing of {cDNA} inserts
from plaques using the linear polymerase chain reaction",
journal = "BioTechniques",
volume = "9",
pages = "48",
year = "1990"}

@article{Rapley1992,
author = "R. Rapley
and S. Flora
and M. R. Walker",
title = "Direct {PCR} sequencing of murine immunoglobulin
genes using {{\em E. coli}} single-stranded {DNA}-binding protein",
journal = "PCR Methods and Applications",
volume = "2",
pages = "99-101",
year = "1992"}

@article{Rosenthal.Charnock-Jones1992,
author = "A. Rosenthal
and D. S. Charnock-Jones",
title = "New Protocols for {DNA} sequencing
with Dye Terminators",
journal = "J. DNA Sequencing and Mapping",
volume = "3",
pages = "61-64",
year = "1992"}

@article{Sanger1977,
author = "F. Sanger
and S. Nicklen
and A. R. Coulson",
title = "{DNA} sequencing with chain-terminating inhibitors",
journal = "Proc. Natl. Acad. Sci. USA",
volume = "74",
pages = "5463-5467",
year = "1977"}

@article{Santillan-Torres1993,
author = "J. L. Santillan-Torres
and P. Ponce-Noyola",
title = "A novel stain for {DNA} in agarose gels",
journal = "Trends in Genetics",
volume = "9",
number = "2",
pages = "40",
comment = "methylene blue, toluidine blue, azure A,
and brilliant cresyl blue",
year = "1993"}

@article{Sharkey1994,
author = "D. J. Sharkey
and E. R. Scalice
and K. G. Christy Jr.
and S. M. Atwood
and J. L. Daiss",
title = "Antibodies as thermolabile switches: high
temperature triggering for the polymerase chain reaction",
journal = "Bio/Technology",
volume = "12",
pages = "506-510",
month = "May",
comment = "antibodies against Taq polymerase; Hot start",
year = "1994"}

@article{Sheng1995,
author = "Y. Sheng
and V. Mancino
and B. Birren",
title = "Transformation of {{\em Escherichia coli}}
with large {DNA} molecules by electroporation",
journal = "Nucleic Acids Res.",
volume = "23",
number = "11",
pages = "1990-1996",
year = "1995"}

@article{Sixou1991,
author = "S. Sixou
and N. Eynard
and J. M. Escoubas
and E. Werner
and J. Teissie",
title = "Optimized conditions for electrotransformation of
bacteria are related to the extent of electropermeabilization",
journal = "Biochimica et Biophysica Acta",
volume = "1088",
pages = "135-138",
year = "1991"}

@article{Solioz1990,
author = "M. Solioz
and D. Bienz",
title = "Bacterial genetics by electric shock",
journal = "Trends in Biochemical Sciences",
volume = "15",
pages = "175-177",
month = "May",
year = "1990"}

@article{Speyer1990,
author = "J. F. Speyer",
title = "A simple and effective electroporation apparatus",
journal = "BioTechniques",
volume = "8",
pages = "28-30",
year = "1990"}

@article{Steele1994,
author = "C. Steele
and S. Zhang
and E. J. Shillitoe",
title = "Effect of different antibiotics on efficiency
of transformation of bacteria by electroporation",
journal = "BioTechniques",
volume = "17",
number = "2",
pages = "360-365",
year = "1994"}

@article{Takahashi1992,
author = "R. Takahashi
and S. R. Valeika
and K. W. Glass",
title = "A simple method of plasmid transformation of
{{\em E. coli}} by rapid freezing",
journal = "BioTechniques",
volume = "13",
pages = "711-715",
year = "1992"}

@article{Taketo1988,
author = "A. Taketo",
title = "{DNA} transfection of {{\em Escherichia coli\/}} by
electroporation",
journal = "Biochimica et Biophysica Acta",
volume = "949",
pages = "318-324",
year = "1988"}

@article{Tang1994,
author = "X. Tang
and Y. Nakata
and H.-O. Li
and M. Zhang
and H. Gao
and A. Fujita
and O. Sakatsume
and T. Ohta
and K. Yokoyama",
title = "The optimization of preparations of competent
cells for transformation of {{\em E. coli}}",
journal = "Nucl. Acids Res.",
volume = "22",
number = "14",
pages = "2857-2858",
comment = "1x10^8 transformants per ug DNA at O.D. 0.94",
year = "1994"}

@article{Thomas1994,
author = "M. R. Thomas",
title = "Simple, effective cleanup of {DNA} ligation
reactions prior to electro-transformation of {{\em E. coli}}",
journal = "BioTechniques",
volume = "16",
number = "6",
pages = "988",
month = "June",
year = "1994"}

@article{Vieira1982,
author = "J. Vieira
and J. Messing",
title = "The {pUC} plasmids, an {M13mp7}--derived system for insertion
mutagenesis and sequencing with synthetic universal primers",
journal = "Gene",
volume = "19",
pages = "259-268",
year = "1982"}

@article{Vogelstein1979,
author = "B. Vogelstein
and D. Gillespie",
title = "Preparative and analytical purification of {DNA}
from agarose",
journal = "Proc. Natl. Acad. Sci. USA",
volume = "76",
number = "2",
pages = "615-619",
month = "February",
comment = "crushed flint glass from scintillation vials used
as Gene Clean binding matrix",
year = "1979"}

@article{Wainwright1993,
author = "L. A. Wainwright
and H. S. Seifert",
title = "Paraffin beads can replace mineral
oil as an evaporation barrier in {PCR}",
journal = "BioTechniques",
volume = "14",
number = "1",
pages = "34-36",
comment = "hot start PCR",
year = "1993"}

@article{Wang1992,
author = "H. Wang
and A. J. Cutler",
title = "A simple, efficient {PCR} technique for
characterizing bacteriophage plaques",
journal = "PCR Methods and Applications",
volume = "2",
pages = "93-95",
year = "1992"}

@article{Welsh.McClelland1990,
author = "J. Welsh
and M. McClelland",
title = "Fingerprinting genomes using {PCR} with
arbitrary primers",
journal = "Nucleic Acids Res.",
volume = "18",
pages = "7213-7218",
year = "1990"}

@article{Williams1990,
author = "J. G. Williams
and A. R. Kubelik
and K. J. Livak
and J. A. Rafalski
and S. V. Tingey",
title = "{DNA} polymorphisms amplified by
arbitrary primers are useful as genetic markers",
journal = "Nucleic Acids Res.",
volume = "18",
pages = "6531-6535",
year = "1990"}

@article{Wirth1989,
author = "R. Wirth
and A. Friesenegger
and S. Fiedler",
title = "Transformation of various species of gram-negative
bacteria belonging to 11 different genera by electroporation",
journal = "Mol. Gen. Genet.",
volume = "216",
pages = "175-177",
year = "1989"}

@article{Woodford1995,
author = "K. Woodford
and M. N. Weitzmann
and K. Usdin",
title = "The use of {K+}--free buffers eliminates a common
cause of premature chain termination in {PCR} and {PCR} sequencing",
journal = "Nucleic Acids Res.",
volume = "23",
number = "3",
pages = "539",
year = "1995"}

@article{Zhixing1995,
author = "Y. Zhixing
and J.-L. Nahon",
title = "{DNA} gyrase improves {DNA} transformation of
{{\em E. coli}} cells with large recombinant plasmids",
journal = "Nucleic Acids Res.",
volume = "23",
number = "16",
pages = "3353-3354",
year = "1995"}

GO BACK TO THE LIST OF QUESTIONS

--

******************************************************************************* * Paul N. Hengen, Ph.D. /--------------------------/* * National Cancer Institute |Internet: pnh@ncifcrf.gov |* * Laboratory of Mathematical Biology | Phone: (301) 846-5581 |* * Frederick Cancer Research and Development Center| FAX: (301) 846-5598 |* * Frederick, Maryland 21702-1201 USA /--------------------------/* * - - - Methods FAQ list -> ftp://ftp.ncifcrf.gov/pub/methods/FAQlist - - - * * - TIBS column archive -> http://www-lmmb.ncifcrf.gov/~pnh/readme.html - - * * - The BEST Molecular Biology HomePage -> http://www-lmmb.ncifcrf.gov/~pnh/ * *******************************************************************************