BAC End-Sequencing

(Diana Bocskai)

1. For every 4 mls of culture, dissolve the BAC DNA pellet in 40 µl of water. for example: Usually each BAC is grown in 20 mls LB/CM total, then is dispensed into one Autogen tube (4 mls in each of the 5 tubes). After miniprep, add 40 µl of water to each tube (200 µl total for each BAC).

    

2. Vortex the Autogen tube and let sit for at least 0.5 hour. Then pool the 5 samples into one for each BAC.

    

3. check the BAC DNA for quality and quantity by digesting 5 µl of the DNA in a 20 µl reaction:

            5.0 µl DNA
            2.0 µl 10x Buffer 2 (NEB)
     0.5 µl Hind III (NEB)
           12.5 µl H2
   O
   

   Digest for 2-4 hours at 37°C.

    

4. Run the digest on a 0.8% agarose gel until the xylene cyanol line is at least 1 inch below the wells. There should be a strong band pattern for each BAC. For example:

    

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5. If DNA is OK for end-sequencing, then prepare 2 reactions for each BAC using T7 and SP6 primers (18mers).

        1 reaction:  22.0 µl DNA
                     16.0 µl reaction mix (ABI/PE #402122)
        2.0 µl 25 µM T7 or SP6
                                   (T7 5'-ATTTAGGTGACACTATAG-3')
                                   (SP6 5'-TAATACGACTCACTATAGGG-3')
   
        PCR conditions: 96°C 4 min.
                        then 25 cycles of:
                        96°C 10 sec.
                        50°C  5 sec.
                        60°C  4 min.
   

6. After PCR, purify samples in columns (Pharmacia #27-5340-03).
   Column protocol:

          1) vortex column;
          2) break off tip at bottom;
          3) place column in eppendorf tube and 
             spin for 1 min. at 3000 rpm;
          4) add all of reaction (40 µl) to top of 
             gel column and place in a new tube;
          5) spin again for 1 min. at 3000 rpm;
          6) speedvac flow-through until all liquid 
             has evaporated;
          7) give dried reaction to sequencing facility.