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| [ 文章来源：www.tree.caltech.edu | 文章作者：
| 发布时间：2007-01-09|
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- 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).
- Vortex the Autogen tube and let sit for at least 0.5 hour. Then pool the 5 samples into one for each BAC.
- 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 H2O
Digest for 2-4 hours at 37°C.
- 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:
- 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.
- 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.
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Overgo Probing of High-D