Berk2010-Daniela

Daniela Mehech 18:58, 7 June 2010 (EDT)

Did the following with Christoph:
Eco/Bam digest of parts: Bjh2245 (LifeACT), Bjh1882 (RFP), and BCA1091 (Ptet)
Gel purified Bjh1882 and Bjh2245
Small fragment zymo cleanup of BCA1091
Couldn't see fragments. Turns out Bjh2245 is 97bp so we should have done a small zymo clean up instead of running it on a gel
Re-cut Bjh2245 and Bjh1882
Did a small zymo clean up of Bjh2245
Did zymo gel purification of Bjh1882 (see image below)

Next we ligated the digested part with the desired vector
Bjh1882+pMLL4 (AC)
Bjh2245+pMLL6 (KA)
Bca1091+pMLL5 (CK)

We don't have pMLL6 in digested form. We'll digest and ligate it tomorrow
We did transformation with Bjh1882+pMLL4 and Bca1091+pMLL5. We transformed them with a Lefty strain of E.Coli (Pir strain, yellow tube) so that the DNA is already methylated

To Do

Digest pMLL6 with Eco/Bam, ligate it with Bjh2245, transform check transformation of Bjh1882 and Bca1091

Daniela Mehech 18:24, 8 June 2010 (EDT)

Still working with Christoph Did EB digest of pMLL6 and did gel purification. Did not take picture of gel because it looked right (a band showed up at approximately 2500)
Ligated pMLL6 digestion with Bjh2245 digestion from yesterday and tranformed into bacteria (not methylated strain, Jtk049)

Colonies appeared from our three transformations (Bjh 1882 with pMLL4and 2 samples of Bca1091 with pMLL5)
We ran a colony PCR on the 3 transformations but the results came out ambiguous (see picture below), so we picked 4 more colonies for more colony PCR
Lanes 1-4 are Bjh1882 and lanes 5-12 are Bca1091

Picked more colonies and ran second gel: Better results this time

To Do

• Miniprep colonies that we pick for pMLL4+Bjh1882 and pMLL5+Bca1091. These are methylated correctly for the robot. Restriction map for confirmation
• Colony PCR pMLL6+Bjh2245 in Jtk049. These are not methylated correctly. Possibly mini-prep if there is enough time

Daniela Mehech 15:33, 9 June 2010 (EDT)

For first Colony PCR of 1882 and 1091: Bca1091 pick lanes 8 and 10 for miniprepping.(Block #1: Colonies from well 1D and 2A)
For second Colony PCR of 1882 and 1091: Bjh1882 pick lanes 1 and 4 for miniprepping. (Block #2: Colonies from well A and D)
Did all 4 mini-preps (stored in working box) and sent in pMLL4-Bjh1882 (D) and pMLL5-Bca1091 (2A) for sequencing

Expected Colony PCR bands:

• pMLL4+bjh1882 = 849bps
• pMLL6+bjh2245 = 265bps
• pMLL5+bca1091 = 228bps
  

Colony PCR of pMLL6+Bjh2245 should be at about 265bps. Looks good. Will mini-prep once culture has grown up