Team:Yale/Our Project/Notebook/Week 8

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iGEM Yale

Monday 7/26--Efforts to confirm that ligation of phsABC and B0015 actually occurred.

Miniprepped the overnight cultures from colonies 5, 12, 14, 15, 20, 21, and 24 according to the vacuum manifold protocol and nanodropped a few to find a ballpark concentration as around 200 ng/uL for each sample.
Based on approximate concentration of miniprepped samples, prepared forward and reverse sequencing reactions for each. Each sequencing reaction mixture had 1.5 uL of the miniprepped DNA, 14.5 uL of water, and 2 uL of the primer, VF2 or VR depending on whether it was a forward or reverse reaction.

This day's work is also recorded on pages 81-82 of the lab notebook hard copy.

While waiting for sequencing data, will digest the miniprepped plasmids of the believed ligation successes with XbaI and SpeI and look to size of resulting fragments as evidence of ligation success.
Ran a digestion of each of the likely ligated plasmids with the following components: 2 uL of NEB 4 buffer, 0.2 uL BSa, 0.7 uL SpeI, 0.7 uL XbaI, 2 uL plasmid DNA, and 14.4 uL of water. Let reaction run for 2 hours at 37˚C followed by a twenty minute heat-kill at 80˚C. Ran on an agarose 1.0% gel at 15 V while out of the lab, but found upon returning that too much of the buffer had evaporated, leading the gel to run extremely crookedly and making it useless.

This day's work is also recorded on page 83 of the lab notebook hard copy.

Reran the double digest of 7/27, but repeating the gel was made unnecessary by the arrival of sequencing data confirming the successful ligation of phsABC into B0015.

>sequence of desired phsABC/B0015 construct

With the phsABC insert finally in B0015, the next step is to insert the Q04121 promoter in front of phsABC. Begin with double digests of Q04121 and the phsABC/B0015 construct double digest
Q04121 double digest: 6 uL of Q04121, 5 uL of EcoRI buffer, 1.8 uL of EcoRI, 1.8 uL of SpeI, 0.5 uL of 100x BSA, and 34.9 uL of water.
phsABC/B0015 construct sequential digest: 5 uL of DNA, 5 uL of NEB buffer 4, 0.5 uL of BSA, 1.8 uL of XbaI, and 37.7 uL of water.
Run both of the digests for six hours at 37˚C before heat-killing with 20 minutes at 80˚C, then add 0.5 uL 5 M NaCl and 1.8 uL of EcoRI to the vector and let it have a five hour digestion period at 37˚C followed by another heat-kill at 80˚C.

This day's work is also recorded on page 83 & 84 of the lab notebook hard copy.

Extra content This day's work is also recorded on page 84 of the lab notebook hard copy.

Extra content This day's work is also recorded on page 85 of the lab notebook hard copy.

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