IGEM:Harvard/2006/Cyanobacteria/Notebook/2006-8-2

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X-S digest of KaiA/B and RFP BioBrick plasmid
We performed X-S digests of KaiA/B and the RFP BioBrick plasmids. There were 6 digests in total: LC3A KaiA, LC3A KaiB, LC3B KaiA, LC3B KaiB, RFP #3, and RFP #4 (RFP numbers refer to the 4 minipreps we did yesterday).

Reaction:
 * 20 µL DNA
 * 2.5 µL Buffer 2 (from NEB)
 * 0.25 µL BSA (100x)
 * 0.5 µL XbaI enzyme
 * 0.5 µl SpeI enzyme
 * 1.25 µL H2O

Incubate for 2 hours at 37C, heat inactivate for 20 minutes at 80C, hold at 4C.

Gel images from yesterday's mutagenic PCR


Gel images from yesterday's "all" PCR


Phosphotase treatment of KaiA/B and RFP BioBrick plasmid digestions
After completing the X-S digests, we added 1 µL alkaline phosphatase to each digestion and incubated for 1 hour at 37C.

Update: This was a mistake, I wasn't supposed to add phosphatase to KaiA and KaiB. We will redo the KaiA/B digests.

Reinoculation of PCC 7942 flask from frozen stock
Our PCC 7942 liquid cultures are now more than 2 weeks old, so we reinoculated a new flask from frozen stock so we have a fresh supply of cyanobacteria.

Gel purification of KaiA/B and RFP BioBrick plasmid
We gel-purified KaiA/B and the RFP BioBrick plasmids after digesting them and treating them with phosphotase. Gel image to the left. 

Gel extraction of BioBrick backbone
We excised the 2kb fragments from lanes 6 and 7 above. They're sitting in the 4C refrigerator waiting to be purified.

Re-digest of KaiA/B and RFP BioBrick
We redigested LC3B KaiA and LC3B KaiB with X-S since I mistakenly added phosphatase to our earlier digests. Also, at Nick's request, we digested the RFP BioBrick again, with the intent of purifying the insert (not the backbone).