Epgfp

BAC Vector for Macro-array Genomic Libraries

Frengen E., Weichenhan D., Zhao B., Osoegawa K., van Geel M., de Jong P. J. 1999. A modular, positive selection bacterial artificial chromosome vector with multiple cloning sites. Genomics. 58(3):250-3. PUBMED 10373322

Abstract

To construct large-insert libraries for the sequencing, mapping, and functional studies of complex genomes, we have constructed a new modular bacterial artificial chromosome (BAC) vector, pBACe3.6 (GenBank Accession No. U80929). This vector contains multiple cloning sites located within the sacB gene, allowing positive selection for recombinant clones on sucrose-containing medium. A recognition site for the PI-SceI nuclease has also been included, which permits linearization of recombinant DNA irrespective of the characteristics of the insert sequences. An attTn7 sequence present in pBACe3.6 permits retrofitting of BAC clones by Tn7-mediated insertion of desirable sequence elements into the vector portion. The ability to retrofit BAC clones will be useful for functional analysis of genes carried on the cloned inserts. The pBACe3.6 vector has been used for the construction of many genomic libraries currently serving as resources for large-scale mapping and sequencing.

NB: pBACe3.6 clones have chloramphenicol antibiotic resistance. Clones should be grown in LB containing 12.5 ug chloramphenicol/ml. Further information on this vector is available from CHORI, Children's Hospital Oakland Research Center

BAC Library Screening

Materials:

Hybridization solution:

• 5x SSPE

• 0.1% NaPPi

• 5% (w/v) SDS

Stripping buffer:

• 0.1x SSC

• 0.1% SDS (w/v)

• 0.2 M Tris-HCl, pH 7.5

DNA Probes:

• See Agilent Prime-II Random Primer Labeling Kit

• Sephadex G50

BAC Miniprep Protocol

This protocol uses alkaline lysis and precipitation to isolate BAC DNA to analyze by Pulsed-field Gel Electrophoresis, PFGE, or PCR. BACs purified using this protocol cannot be injected into fertilized eggs.

Materials:

Buffer P1: Stored at 4^oC. Add the RNAseA just prior to use.

• 15 mM Tris, pH 8.0

• 10 mM EDTA, pH 8.0

• 100 μg/ml RNase A

Buffer P2: Make fresh each use.

• 0.2N NaOH

• 1% SDS

Buffer P3: Cool on ice prior to use.

• 3M KAc pH 5.5

Procedure:

1. Inoculate a single bacterial colony into 3 ml LB containing 12.5μg/ml chloramphenicol in a 14 ml culture tube. Grow overnight (< 16 hrs), shaking at 250-300 rpm.

Optional: make bacterial glycerol stock (15%) of BAC.

2. Pellet the bacteria by transferring 1.5 ml of each culture to a 1.7 ml microcentrifuge tube and centrifuge at 6800 g for 3 min. Discard supernatant.

3. Repeat step 2.

4. Resuspend each pellet in 250 μl P1 carefully. Be sure to fully resuspend until suspension is creamy with no clumps.

5. Add 250 μl P2 and invert tubes 5 times to mix. The appearance of the suspension should change from very turbid to almost translucent.

6. Add 350 μl cold P3 slowly to each tube and shake gently during addition. A thick white precipitate consisting of E. coli DNA and protein will form. Invert the tube several times to mix the solution thoroughly.

7. Place the tubes on ice for 5 min.

8. Centrifuge at 18,000 x g for 10 min at room temperature to pellet the white precipitate.

9. Transfer the clear supernatant (~700-800 μl) to a 1.7 ml microcentrifuge tube.

10. Spin again in a microcentrifuge for 5 min at RT to remove the rest of the debris. Transfer the clear supernatant to a fresh tube.

11. Add 0.8 ml ice-cold isopropanol. Mix well by inverting tubes ~10 times. Place the tube on ice for 30 min, or leave overnight at 4°C.

12. Centrifuge at 18,000 x g for 30 min at 4^oC to pellet BAC DNA.

13. Remove supernatant and add 1ml of ice-cold 70% EtOH. Invert tubes several times to wash the DNA pellets. Centrifuge at 18,000 x g for 15 min at 4^oC.

14. Repeat step 13.

15. Centrifuge at 18,000 x g for 2 min at 4^oC to remove residual EtOH. Carefully remove all supernatant, taking care not to dislodge the pellet.

16. Briefly air-dry pellet at room temperature.

17. Resuspend pellet in 20-30 μl TE (10 mM Tris; 1 mM EDTA). Gently flick the bottom of the tubes to resuspend DNA. Do not vortex or pipet up and down.

For storing use high EDTA TE - i.e. 10mM Tris 10mM EDTA.

To analyze the BACs, use 6 μL of this prep in a NotI digest to run on a PFGE.

For PCR dilute 1 μl of this prep in 24 μl TE.