Berk2010-Christoph: Difference between revisions
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*Sent in miniprepped pMLL4-Bjh1882 (D) and pMLL5-Bca1091 (2A) for sequencing. | *Sent in miniprepped pMLL4-Bjh1882 (D) and pMLL5-Bca1091 (2A) for sequencing. | ||
*Started growing up Lefty and Righty strains. Picked two colonies of each lefty and righty and grew them up in 10ml LB. | |||
==[[User:Christoph Neyer|Christoph Neyer]] 13:28, 9 June 2010 (EDT)== | ==[[User:Christoph Neyer|Christoph Neyer]] 13:28, 9 June 2010 (EDT)== | ||
Revision as of 12:41, 9 June 2010
Christoph Neyer 15:16, 9 June 2010 (EDT)
Colony PCR of pMLL6+Bjh2245 should be at about 265bps. Looks good.
- Sent in miniprepped pMLL4-Bjh1882 (D) and pMLL5-Bca1091 (2A) for sequencing.
- Started growing up Lefty and Righty strains. Picked two colonies of each lefty and righty and grew them up in 10ml LB.
Christoph Neyer 13:28, 9 June 2010 (EDT)
Bca1091 is 60bps.
Colony PCR band for parts:
- pMLL4+bjh1882 = 849bps
- pMLL6+bjh2245 = 265bps
- pMLL5+bca1091 = 228bps
- For First try Colony PCR of 1882 and 1091:
- Bca1091 pick lanes 8 and 10 for miniprepping.(Block #1: Colonies from well 1D and 2A)
- For Second try Colony PCR of 1882 and 1091
- Bjh1882 pick lanes 1 and 4 for miniprepping. (Block #2: Colonies from well A and D)
Christoph Neyer 19:37, 8 June 2010 (EDT)
TO DO Tomorrow:
- Miniprep colonies that we pick for pMLL4+Bjh1882 and pMLL5+Bca1091. These are methylated correctly for the robot.
- Colony PCR pMLL6+Bjh2245 in Jtk049. These are not methylated correctly.
Christoph Neyer 19:51, 8 June 2010 (EDT)
- Ran gel of colony PCR pMLL4-Bjh1882 and pMll5-Bca1091 AGAIN:
| ladder | 1882a | b | c | d | 1901a(1) | b | c | d | ladder | 1901a(2) | b | c | 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.
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 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 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 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.
