PREPARATION OF SINGLE STRANDED UTP+ DNA FOR MUTAGENESIS
1. Subclone your insert of interest into a plasmid that contains an M13 intergenic sequence; CDM8, Bluescript and AprM8 work well. Be sure you know which strand will be rescued by the helper phage, so that the mutagenic oligonucleotide will be synthesised complementary to the ssDNA. (In CDM8 and AprM8 containing an insert with the 5' end adjacent to the CMV promoter, the rescued strand is the antisense strand, so synthesize primers corresponding to the strand representing the mRNA sequence).
2. Transform your plasmid into competent BW313/p3 (or CJ236 for AprM8 or BSII). Plate the transformation mixture on Amp/Tet plates for BW313 derivatives) and Amp for CJ236. Note always grow CJ236 (and subsequent plasmid transformants) in the presence of 30ug/ml chlorampheriol (30mg/ml stock in ethanol) as it's F pilus is easily lost without selection. To be sure that all is well, pick a few colonies, miniprep the plasmid and digest it with enzymes that give characteristic patterns for your insert on agarose gel eletrophoresis. Make a glycerol stock of the BW313/p3 containing your plasmid for your fututre use.
3. Inoculate 4ml overnight culture of BW313/p3 or CJ236 containing your plasmid in LB/Amp/chloramph.
4. The following afternoon, inoculate a 220 ml culture of LB/Amp/Chloram. with 4ml of the fresh overnight culture. Allow to grow to an OD600 = 0.1. Assuming that 1 OD600 = 8 x108 cfu/ml, add 10-12 fold excess of M13KO7 helper phage and shake overnight.
5. When the culture is done, pellet the bacteria by centrifugation at 12,000 rpm for 15 min in the GSA rotor using the 250ml bottles. Transfer the supernatant to a fresh bottle and recentrifuge to completely remove bacteria and particulates (do not skip this!). Transfer 200ml of the supernatant containing the phagemids to a fresh bottle and add 10ul of 10mg/ml RNaseA, swirl and let sit on the benchtop for 30 min. Add 50ml of 20% PEG(8000)/2.5M NaCl and incubate in ice water for 1-2 hr. Filter the remaining 20ml of phage through a 0.45m filter and store at 4¡C. This can be used to check for uracil incorporation.
6. Pellet the precipitated phagemids by centrifugation at 12,000 rpm for 15 min, note the orientation of the bottle in the centrifuge. The phagemids (brown in colour) will form a small pellet and streak up the side of the tube. Re-centrifuge 12,000rpm for 5 min again noting the orientation of the bottle. The phagemids will tend to form a tighter pellet at the bottom of the tube. Remove all traces of PEG by suction with a Pasteur pipette. Resuspend the phagemids in 1ml of STE (20 mM Tris-HCl pH 8.0/50mM NaCl/2 mM EDTA). Split the sample into two eppendorf tubes. At this point the phagemids can be stored at 4¡C for up to 1 week.
7. Add an equal volume of Tris-saturated phenol to each tube, vortex for 30 sec, allow to stand for 30 sec, and re-vortex for 30 sec. Separate phases by microcentrifuging at 14,000g for 5 min. Transfer the upper aqueous phase to a fresh tube and extract with phenol:CIAA. Centrifuge for 2 min, transfer the upper aqueous phase to a new tube and re-extract with phenol:CIAA. Extract once with CIAA alone.
8. Add 1/10 volume 3M sodium acetate pH 5.2 and 2 volumes of ethanol, mix and incubate at -70¡C for 30 min. Thaw, and microfuge for 10-15 min at 14,000g. The DNA will be a fairly large white pellet streaked up the side of the tube. Remove ethanol, dry and resuspend in 50ul of TE. Analyse and estimate the concentration of the DNA by agarose gel electrophoresis using ssM13 as a control. The DNA is ready to be used for mutagenesis.
(Note: to prepare additional helper phage, substitute Ecoli strain MV1190 without a plasmid for BW313/p3 above. In the absence of a plasmid to package, M13KO7 will package itself. Proceed through step 6 but resuspend the phage in 5-6ml of STE and filter through a 0.45um filter. Store at 4¡C, or add DMSO to 8% and freeze. The helper phage may be titered on a lawn of MV1190 by serial dilution, as usual).
Checking uracil incorporation:
100ul of an O/N culture of BW313/p3 (dut - ung -)+ 0
+ 5ul of S/N containing UTP+ phage
100ul of an O/N culture of MC1061/p3 (dut+ ung+)+ 0
+ 5ul of S/N containing UTP+ phage*
Incubate at 37¡C for 29 min to allow phage to absorb to bacteria. Plate on LB/Amp/Tet and grow overnight at 37¡C. *Relatively few colonies should be recovered following infection of dut+ung+ strain with uracil-containing phage DNA.
2. PHOSPHORYLATION OF OLIGONUCLEOTIDES
1. Oligonucleotides are synthesised with 5'-hydroxyl ends. T4 DNA ligase requires 5' phosphate ends in order to ligate DNA efficiently. Thus synthetic linker or oligonucleotides for mutagenesis studies be phosphorylated before use.
5ug purified oligonucleotide
7.5ul 1M Tris-HCl pH7.5
1ul 1/10 dilution of 2-mercaptoethanol
7.5ul 0.1M MgCl2
1ul 100mM ATP
2ul T4 polynucleotide kinase
H20 to 75ul
Incubate 2hr - overnight at 37¡C
3. MUTAGENESIS REACTIONS
500ng single stranded UTP+ DNA
266ng phosphorylated oligonucleotide
H20 to 13ul
90¡C for 5 min to denture oligonucletode
37¡C for 10 min to anneal oligonucleotide to template
0¡C to stabilize
4ul 5 X Sequenase buffer (United States Biochemical)
1ul second strand additions (*see below)
1ul T4 DNA ligase
1ul T7 DNA polymerase (Sequenase; United States Biochemical version 1)
25¡C for 5 min to generate second strand
37¡C for 90 min to anneal
Transform high efficiency competent bacteria with 2.5ul of mutagenesis reaction mix and plate the lot on antibiotic selection plates. Grow overnight at 37¡C. Freeze remaining reaction mix at -20¡C. Generation of closed circular DNA can be monitored by gel electrophoresis.
*Second strand additions (for 20ul)
13mM dNTP (10ul 25mM dNTP; Ultrapure dNTP set, Pharmacia LKB Biotechnology, Catalouge #27-2035-01)
10mM ATP (4ul 50mM ATP; Adenosine 5'-triphosphate disodium salt, Sigma, Catalogue #A-6144)
30mM DTT (3ul 200 mM DTT)