BAC modification

From Genome@Yale

BACPAC Resources Center (BPRC)

Contents 1 lambda recombinase mediated modification 2 BAC Purification 2.1 BAC miniprep 2.2 BAC restriction digest 3 Transformation 3.1 Electrocompetent cells 3.2 Electroporation

lambda recombinase mediated modification

NCIFCRF BAC recombineering protocols:

• BAC Protocol 1
• BAC Protocol 2
• BAC Protocol 3
• BAC Protocol 4

BAC Purification

Nucleobond BAC 100 kit from Clontech (catalog number 635941) for a large prep

BAC miniprep

Use Qiagen miniprep buffers.

1. Using a sterile toothpick, inoculate a single isolated bacterial colony into 2 ml 2xYT (or LB) media supplemented with 12.5ug/ml chloramphenicol for BAC clones or with 25 ug/ml kanamycin for PAC clones. Use a 12-15 ml snap-cap polypropylene tube. Grow overnight (up to 16 hours) shaking at 225-300 rpm at 30C.
2. Transfer the culture to 2mL eppendorf tubes. Centrifuge 15000 rpm for 2.5 minutes.
3. Aspirate supernatants. Resuspend each pellet in 0.25 ml P1 solution. Add 0.25 ml of P2 solution and gently shake tube to mix the contents. Let sit at room temperature for 5 minutes. The appearance of the suspension should change from very turbid to almost translucent.
4. Slowly add 0.3 ml P3 solution to each tube and gently shake during addition. A thick white precipitate of protein and E. coli DNA will form. After adding P3 solution to every tube, place the tubes on ice for at least 5 min.
5. Centrifuge 15000 rpm for 10 minutes at 4C.
6. Remove tubes from centrifuge and place on ice. Transfer supernatant to a 1.5 ml eppendorf tube that contains 0.67 ml ice-cold isopropanol. Try to avoid any white precipitate material. Mix by inverting tube a few times; place tubes on ice for at least 5 minutes. At this stage, samples can be left at -20C overnight.
7. Spin in cold microfuge for 15 minutes.
8. Remove supernatant and add 0.5 ml of 70% EtOH to each tube. Invert tubes several times to wash the DNA pellets. Centrifuge for 5 minutes. Optional:repeat wash.
9. Remove as much of the supernatant as possible. Occasionally, pellets will become dislodged from the tube so it is better to carefully aspirate off the supernatant rather than pour it off.
10. Air dry pellets at room temp. When the DNA pellets turn from while to translucent in appearance, i.e., when most of the ethanol has evaporated, resuspend each in 50 ul 10mM Tris, pH 8.0. Do not use a narrow bore pipets tip to mechanically resuspend DNA sample; rather, allow the solution to sit in the tube with occasional tapping of the bottom of the tube. For large PAC clones resuspension may take over 1 hour.
11. Use 5 uL for digestion with NotI or other rare cutter enzymes. There are Notl sites flanking the Sp6 and T7 promotor regions of the CYPAC2 vector; therefore, this is a very useful enzyme for analysis of insert size and for partial digest restriction mapping. Use 10 uL for digestion with a more frequent cutter such as BamHI or EcoRI.

BAC restriction digest

1. Make mastermix (2uL 10X NEB3, 1uL BamHI, 7uL dH2O) and transfer 10uL to new tube
2. Add 10uL BAC miniprep DNA and mix
3. Incubate 37C for 2 hour
4. Heat inactivate enzyme at 65C for 10 minutes
5. Run 0.8% agarose, 1.5 hours at 90V

Transformation

Prepare BAC miniprep DNA and electrocompetent cells the same DNA.

Electrocompetent cells

1. Seed 2mL 2xYT media with SW102 or derivatives for overnight.
2. Dilute cultures 0.5mL into 25mL 2xYT (50mL conical tube) grow until OD ~0.6
3. Chill the cultures on ice
4. Centrifuge 6000 rpm 5 minutes 4C
5. Discard supernatant
6. Gently resuspend pellet in 1mL dH2O by swirling the tube in ice water, then add 9mL dH2O, mix by swirling
7. Centrifuge 6000 rpm 5 minutes 4C
8. Discard supernatant
9. Gently resuspend pellet in 1mL dH2O by swirling the tube in ice water, then add 9mL dH2O, mix by swirling
10. Centrifuge 6000 rpm 5 minutes 4C
11. Discard supernatant
12. Use residual dH2O to resuspend the pellet

Electroporation

Chill everything on ice

1. Mix 5uL BAC DNA with 20uL electrocompetent cells
2. Transfer mixture to cuvette (carefully)
3. Zap the cells
4. Add 1mL LB and transfer the culture to Eppendorf to tube
5. Recover ~1 hour at 30C
6. Spin down cells and plate all the cells onto LB + CM plate
7. Incubate at 30C O/N
8. Pick colonies, miniprep and fingerprint to make sure no large recombinations