Berk2010-Daniela

Daniela Mehech 19:04, 11 June 2010 (EDT)

Colonies grew from pMLL5+Bca1152 so I ran a colony PCR and inoculated 4 cultures. From colony PCR (band at 250), decided to miniprep colonies C and D. Sent D to sequencing, added it to parts plate (source) and transformation plate (destination). Mini prep from C went to Working Box

Added grown up Lefty and Righty strains to 500ml each of LB. Tim made competent cell stock

Finished with Christoph our CVS file for robot to dilute our parts in a new plate. Used robot to transform all of our plasmids into the right Righty/Lefty strain. Plated everything. Someone will move the plates from the incubator to the fridge tomorrow

Daniela Mehech 14:38, 10 June 2010 (EDT)

Sequences for all parts we made were good (pMLL4+Bjh1882, pMLL5+Bca1091, pMLL6+Bjh2245)
Our competent cell cultures did not grow up at all - inoculated 2 more cultures of each strain and they appear to be growing well
Christoph and I double checked to confirm we had all the parts we need in the right vector - we're missing pMLL5+B10sbb37/Bca1152)
We took the part from the 140L stock, did a E/B digest, ligated it with pMLL5 digest and transformed it into jtk049 strain
I updated the pictures of the assembly tree
Began planning to do transformations of all our parts into righty and lefty strains

To Do

Pick colonies from Bca1152 transformation for colony PCR. Plate colonies in both CK plate and Spec plate. Nothing should grow in Spec plate
Make competent Pir cells
Transfer parts from working box to PCR plate
Write file for robot to do our transformations (first make dilutions from PCR plate)
When oligos come in - Biobrick upstream region of ef1-alpha

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-mini-preped lanes 2 and 3 and sent both for sequencing

Started growing up Lefty and Righty strains. Picked two colonies of each lefty and righty and grew them up in 10ml LB.
Designed three potential oligos for biobricking part Bjh2341CA (igemTen011, igemTen012, and igemTen013)
Bjh2341CA is a potential choano promoter (its a DNA region about 1kb upstream of ef1-alpha, a highly expressed gene)

TO DO

• Finish generating competent cells for "Lefty" and "Righty" strains.
• Analyze sequencing data for pMLL6+Bjh2245, pMLL4+Bjh1882 and pMLL5+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 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