Berk2010-Christoph: Difference between revisions
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Revision as of 15:15, 8 June 2010
Christoph Neyer 15:54, 7 June 2010 (EDT)
Eco/Bam digest of parts: Bjh2245 (LifeACT), Bjh1882 (RFP), and BCA1091 (Ptet).
Gel purified Bjh1882 and Bjh2245.
Small fragment zymo cleanup of BCA1091.
Christoph Neyer 16:35, 7 June 2010 (EDT)
Couldn't see fragments. Turns out Bjh2245 is 97bp and Bjh1882 is ~680bps.
Re-cut Bjh2245 and Bjh1882.
Small fragment zymo cleanup of Bjh2245 digest.
Gel purified Bjh1882.
Christoph Neyer 19:30, 7 June 2010 (EDT)
Bjh1882 Vectors: AC
Bjh2245 Vectors: KA
Bca1091 Vectors: CK
pMLL4-AC
pMLL5-CK
pMLL6-KA
pMLL7-AK
pMLL8-KC
pMLL9-CA
Bjh1882+pMLL4-AC = Lefty
Bjh2245+pMLL6-KA = Righty
Bca1091+pMLL5-CK = Lefty
Ligated isolated Bjh1882 and Bca1091 into above vectors. Transformed into Lefty strains.
Need to ligate Bjh2245 (Missing pMLL6, need to digest).
TO DO:
- Digest pMLL6 with Eco/Bam and gel purify. Then ligate with Bjh2245.
- Check transformed Bjh1882 and Bca1091.
- Transform ligated Bjh2245+pMLL6 into Righty strain
Christoph Neyer 14:04, 8 June 2010 (EDT)
- Digested pMLL6 w/ Eco/Bam and gel purified.
- Colony PCR of transformed pMLL4-Bjh1882 and pMLL5-Bca1091:
| PCR Block 1882 and 1091 #1 | ' | ' | ' | ' |
| 1882 | 1091 #1 | 1091 #2 | ||
| A | A | A | ||
| B | B | B | ||
| C | C | C | ||
| D | D | D |
Christoph Neyer 16:59, 8 June 2010 (EDT)
- Ligated pMLL6 Eco/Bam digest with part Bjh2245.
- Ran gel of colony PCR pMLL4-Bjh1882 and pMll5-Bca1091:
| ladder | 1882a | b | c | d | 1901a(1) | b | c | d | ladder | 1901a(2) | b | c | d |
- Repicking colonies since Colony PCR appeared contaminated. Block has same layout, shown below:
| PCR Block 1882 and 1091 #2 | ' | ' | ' | ' |
| 1882 | 1091 #1 | 1091 #2 | ||
| A | A | A | ||
| B | B | B | ||
| C | C | C | ||
| D | D | D |
- Ran out of "Righty" cells. So we are transforming Jtk049 with our E/B ligation of pMLL6+Bjh2245.
