A. BAC Vector Preparation (Non-CsCL)
B. BAC Vector Preparation (CsCl)
E. Colony Filters for Library Screening
pBeloBAC11 was developed by Drs. M. Simon and H.Shizuya (unpublished; Shizuya et al, 1992). pBeloBAC11 represents the second generation BAC cloning vectors, which wasdeveloped from the pBAC108L (Shizuya et al 1992) by introducing the LacZgene to facilitate recombinant identification with blue and colorless(white) phenotypes. pBeloBAC11 is a mini-F factor based plasmid. There arethree unique cloning sites: Bam HI, SphI, and Hind III, which are flankedby the T7 and SP6 promoters. These promoters can facilitate generating RNAprobes for chromosome walking and DNA sequencing of the insert fragment atthe vector - insert junction. The G + C rich restriction sites (Not I, EagI, Xma I, Sma I, Bgl I, and Sfi I) can be used to excise the inserts of BACclones. There are two selective markers for cloning purposes: LacZ gene forrecombinant selection and CMR (chloramphenicol) for transformant selection.The F factor codes for genes that are essential to regulate its ownreplication and controls its copy number in a cell. The regulatory genesinclude oriS, repE, parA, and parB. The oriS and repE mediate theunidirectional replication of the F factor, and the parA and parB maintaincopy number at a level of one or two per cell.
b. Inoculate a single colony in 5 ml TB medium plus 12.5 mg/mlchloramphenicol and grow at 37°ree;C for 6 - 8 hours with shaking at 250 rpm.
c. Inoculate 1 ml of the culture into 1 liter of TB 12.5 mg/mlchloramphenicol and grow at 37°ree;C overnight with shaking at 250 rpm.
d. Harvest the cells in five 250 ml centrifuge bottles by centrifugationat 8,000 rpm (~ 10,000 g) for 10 minutes and pour off the supernatantfluid.
e. Resuspend the cell pellet in each bottle in 20 ml of lysozyme solutionby repeatedly pipetting up and down and incubate on ice for 5 minutes.
f. Add 40 ml of NaOH-SDS solution to each bottle, mix gently, and incubateon ice for 5 minutes.
g. Add 30 ml of ice cold KOAc solution and mix gently. A white precipitateshould form. Freeze the mixture in a - 80°ree;C freezer until mixture iscompletely frozen (~ 30 minutes) and then let thaw at room temperature.
h. Centrifuge at 10,000 rpm (~ 15,000g) for 15 minutes to collect theprecipitate and filter the supernatant into a clean 250 ml centrifugebottle through 4 layers of cheese cloth.
i. Add 0.6 volume of isopropanol to the supernatant fluid, mix well, andfreeze the contents as in step 7 and then warm to room temperature.
j. Centrifuge at room temperature at 10,000 rpm for 30 minutes to pelletthe DNA, remove the supernatant, and rinse the pellet with 50 ml 70%ethanol.
k. Centrifuge at 10,000 rpm for 15 minutes, remove the 70% ethanol, dry,and dissolve the DNA in 1 ml TE (10 mM Tris.HCl, pH 8.0, 1 mM EDTA, pH8.0).
l. Combine all the DNA isolated from 1 liter of culture into one clean 15ml centrifuge tube.
m. Add DNase free RNase at a rate of 20 mg/ml and incubate at 37°ree;C for 45minutes to remove the RNA in the DNA.
n. Extract the DNA with an equal volume of equilibrated phenol once and 25phenol : 24 chloroform : l isoamyl alcohol once.
o. Precipitate the DNA by adding 1/10 volume of 3M NaOAc and 2 volumes ofethanol, dry, and dissolve in 200 m1 TE (the concentration of DNA should be0.5 - 1 mg/ml).
a. Set up the digestion as follows:
b. Incubate the reaction at 37°ree;C for 2 hours.
c. Add an additional 4 ml restriction enzyme and incubate for additional2 hours.
d. To ensure that the digestion is indeed complete, test the digestion onan 1% agarose gel and/or by transformation into E. coli using the uncutplasmid DNA as a control (circular plasmid DNA molecules should be < 20%)
. e. Extract the digest once with phenol: chloroform (1:1), precipitate,dry, and dissolve in H2O.
f. Set up the following reaction to dephosphorylate the ends of linearized vector DNA:
g. Incubate at 37°ree;C for 30 minutes and stop the reaction by adding 5 mMEDTA, pH 8.0, 0.5% SDS , and 0.1 mg/ml proteinase K and incubating at 56°ree;Cfor 30 minutes.h. Extract twice with phenol, once with phenol : chloroform : isoamyalcohol (25:24:1), precipitate with ethanol, dry, dissolve in H2O at 10ng/ml and store in 10 ml aliquots at - 80°ree;C.
Note: A preliminary experiment is often performed to optimizedephosphorylation conditions. A large scale of digested vector DNA isdephosphorylated under the optimal conditions.
a. Streak HS995 cells on an LB plate containing CM (30 mg/ml), X-GAL (240ml of 20 mg/ml) and IPTG (24 ml of 200 mg/ml) and grow at 37°ree;C overnight.
b. Inoculate a single blue colony in 4 L of LB media, prewarmed at 30°ree;C,containing 30 mg/ml CM.
c. Grow the inoculum 20 hours at 30°ree;C to an OD600nm between 1.0 and 1.5,and then harvest the cells in eight 500 ml centrifuge bottles bycentrifugation at 6000xg for 15 minutes.
d. Remove all the traces of supernatant by inverting the centrifugebottles for a few minutes. Isolate plasmid DNA from the cell pellet by alkaline lysis using theQIAGEN plasmid Maxi Kit according to manufacturer's specifications withmodifications (Five QIAGEN tip 500s can be used for a 4 L preparation).
e. Resuspend the bacterial pellet in each bottle completely with 10 mlbuffer P1.
f. Transfer the bacterial solution to eight Oakridge tubes.
h. Add 10 ml buffer P2, mix gently by inverting several times and incubateat room temperature for 5 minutes.
i. Add 10 ml prechilled buffer P3 on ice, mix immediately but gently, andincubate on ice for 20 minutes.
j. Centrifuge at 30,000xg at 4°ree;C for 30 minutes, remove the supernatant tonew Oakridge tubes and repeat this step.
k. Equilibrate five tip 500s with 10 ml buffer QBT.
l. Apply approximately 50 ml of the supernatant from step 9 to each 500tip, and allow it to enter the resins by gravity flow.
m. Wash the tips twice each time with 30 ml buffer QC.
n. Elute DNA with 15 ml buffer QF.
o. Precipitate DNA with 0.7 volume of ice-cold isopropanol and keep at-20°ree;C overnight.
p. Centrifuge at 30,000xg, remove the supernatant, and air-dry the pellets.
q. Resuspend the pellets in TE, and combine the DNA solution to one 15 mltube in a final volume of 5ml. Precipitate the DNA again with 0.1 volume of3M NaAc, pH 5.2 and two volumes of ethanol.
r. Wash the pellet carefully with ice-cold 70% ethanol, air-dry, andresuspend in 1 ml of TE.
s. Purify the plasmid DNA further by cesium chloride density gradientcentrifugation in the presence of ethidium bromide.
i. Prepare a CsCl2 gradient with a density of 1.59 g/ml with EtBr at 0.8 mg/ml.
v. Dilute the DNA sample five fold with TE, and then precipitate withethanol and centrifuge. Wash the pellet with ice-cold 70% ethanol, air-dryand resuspend in 500 ml of TE. The final yield should be approximately50-70 mg of pBeloBAC 11 DNA from 4 L of media.
b. Incubate the reaction at 37°ree;C for 5 h.
c. Verify the complete digestion on an agarose gel with the uncut DNA.
d. Extract the DNA with phenol followed by 2 chloroform extractions.
e. Precipitate the DNA with ethanol as above and resuspend in 100 ml ofTE. Save 1 ml for a control of self-ligation test.
f. Set up the dephosphorylation reaction as follows:
g. Inactivate the HK phosphatase by heating at 67°ree;C for 30 minutesfollowed by the organic extractions and an ethanol precipitation as aboveand resuspend in TE in a final concentration of 0.1 mg/ml.h. Assay the extent of dephosphorylation by performing a self ligationtest as follows:
i. Incubate the ligation reactions overnight at 16°ree;C, and verify thecomplete dephosphorylation on an agarose gel with the uncut DNA. Store onemg aliquots of the dephosphorylated pBeloBAC 11 plasmid at -80°ree;C untilneeded.
a. After ligation, transfer the ligation mixture with a cut off pipet tipto a Millipore filter unit which is placed in a 1.5 ml microfuge tubecontaining approximately 1 ml of TE at 4°ree;C to dialyze out the ligationbuffer.
b. Change the dialysis solution with 0.1x TE two additional times over a24 hour period.
c. Transform E. coli ElectroMAX DH10B cells (BRL, USA) by electroporationusing the BRL Cell-Porator system with the manufacturer's transformationprocedure.
d. Place micro-electroporation chambers and 0.5 ml microcentrifuges tubeson ice. Fill Cell-Porator Chamber-SafeTM with wet ice.
e. Place 1 ml S.O.C. medium in 15 ml sterilized culture tubes.
f. Add 1 ml of dialyzed ligation material to a 0.5 ml microfuge tube on ice.
g. Take out competent cells from -80°ree;C and thaw on ice. Add 20 ml cells toeach microcentrifuge tube, and gently mix them by tapping.
h. Pipet the mixture of DNA and cells into micro-electroporation chambers.
i. Electroporate with the BRL cell-Porator and Voltage Booster at thefollowing settings:
j. Remove the transformed cells from the micro-electroporation chamber andresuspend in 1 ml of SOC medium and incubate at 37°ree;C with shaking at 225rpm for one hour to allow expression of the CM resistant gene.
k. Plate 250 ml of the cells per LB plate (150x15 mm) containing CM,X-GAL, and IPTG.
l. Incubate for 18 to 24 h at 37°ree;C.
NaOH-SDS stock: 0.2 N NaOH, 1% SDS. Prepare fresh by mixing equal volumes of 0.4 N NaOH and 2% SDS.
Potassium acetate stock: 60 ml 5 M potassium acetate, 28.5 ml glacialacetic acid, 11.5 ml ddH2O, pH 4.8
a. Prepare a 5 ml culture of LB medium containing 12.5 mg/ml chloramphenicol.
b. Inoculate a single colony and incubate with shaking at 37°ree;C for 18 - 20 h.
c. Centrifuge the overnight culture at 3000 rpm (approximately 1500g) at4°ree;C for 15 minutes using a table top centrifuge (Beckman).
d. Pour off the supernatant fluid and resuspend the cell pellet in 0.2 mlof lysozyme solution. Transfer to a 1.5 ml microfuge tube and incubate for5 minutes at room temperature and five minutes on ice.
e. Add 0.4 ml of NaOH-SDS stock and invert gently. The solution shouldturn translucent. Incubate for 5 minutes on ice.
f. Add 0.3 ml of the potassium acetate stock and invert gently. A whiteprecipitate should form. Freeze at -80°ree;C for 10 - 15 min. Let thaw at roomtemperature.
g. Centrifuge for 15 minutes in a microfuge (approximately 12,000g) tocollect the precipitate.
h. Carefully remove as much the supernatant fluid as possible (about 0.85ml) without disturbing the pellet and transfer to a clean microfuge tube.
i. Recentrifuge for 5 minutes if necessary and remove the supernatant as above.
j. Add 0.6 volumes of ice cold isopropanol (usually 0.54 ml) and gentlymix. Freeze at -80°ree;C for 10-15 minutes. Warm to room temperature andcentrifuge for 20 minutes in a microfuge 4°ree;C to pellet the DNA.
k. Remove the supernatant fluid and rinse the pellet with 1 ml of ice cold70% ethanol. Centrifuge for 2 minutes and pour off the ethanol rinse anddry the tubes upside down. Air-dry for 10-15 minutes.
l. Add 40-50 ml of TE and resuspend the pellet at 65°ree;C for 5 minutes andcentrifuge briefly.
m. Digest 10 ml of DNA with NotI to free the insert from the BAC vector.
On ice, prepare a cocktail with 20 x 9.5 ml H2O, 20 x 2.5 ml 10 xreaction buffer, 20 x 2.5 ml 40 mM spermidine, and 20 x 0.5 ml NotI.Aliquot 15 ml of the cocktail to each tube containing the 10 ml of BAC DNA.(A single restriction digestion has the following composition.) BAC DNA 10 ml 10X NotI reaction buffer 2.5 ml SPD (40mM) 2.5 ml ddH2O 9.5 ml Not I 0.5 ml Total 25 ml
2. Inoculate each filter with 96 BAC clones using a 96 well replicaplating device. Repeat this step 3 additional times for a single filter ifprocessing by hand or 16 to 36 times if using a robot (BIOMEK 2000, BeckmanUSA).
3. After inoculating all of the filters, incubate the plates forapproximately 18 to 24 hours at 37°ree;C.
4. One hour before processing the colonies prepare the following trays.
Place 6 developing trays side by side, label in the order as above. Cutand lay Whatman filter paper in the bottom of each tray (1 sheet per tray).Pour approximately 50 ml of each solution into the appropriate tray androll out the air bubbles with a Pasteur pipet. Pour the excess solutionback into its original container for use later.
5. Carefully remove 3 filters with a flat edged forceps and place themonto the 10% SDS tray. Incubate the filters for 4 minutes.
6. After 4 minutes move the filters to the 2nd tray containing theNaOH/NaCl solution and incubate for 5 minutes.
7. Transfer the filters to the neutralizing solution (0.5 m NaOH, 1.5 mNaCl) and incubate for 5 minutes.
8. Wash the filter for 5 minutes with 2 X SSC and 1% SDS. Then, wash for 5minutes with 2 X SSC without SDS.
9. Fix the DNA to the filter with a 20 minute incubation with 0.4 N NaOH.
10. Thoroughly wash the filters two times for 20 minutes each with a largevolume of 5 X SSC + 0.1 % SDS with shaking.
11. Once the cell debris is removed, wash the filters with two 5 minuteschanges of 2 X SSC. The filters are now ready for pre-hybridization.