AUGUST

Friday, August 1

Discussed with Sun to troubleshoot why the PCR wasn’t working. We discovered that the final base in one of our primers was incorrect. We corrected that and reordered the primer.

Tuesday, August 5

Reran PCR with correct primer using previously isolated E. coli genome.

Wednesday, August 6

Checked PCR with gel, this gel failed. Reran PCR using a gradient of temperatures. Checked this PCR with a gel, which also failed.

Thursday, August 7

Reran Gel with higher concentrations of PCR product. This gel also failed.

Friday, August 8

Discussed with Sun why the PCR was not working. Discovered that when finding the melting temperatures we had not used the specifications of the polymerase we were using. Reran the PCR using a gradient around the new melting temperature. We used both colonies and previously isolated E. coli genome in different replicates for our template DNA. Ran a gel to determine success. The conditions which gave us the required 1.7 kb strand were 61 ºC and 1.7 minute extension step for the first five cycles and 72 ºC and 1.7 minute extension step for the last 30 cycles. The template DNA that worked came from the previously isolated E. coli genome.


Gel Results, August 8

Monday, August 18

Performed restriction enzyme digest on the PCR product (FadD) and the previously minipreped lac promoter from the 2014 distribution kit plate 3 well 4G, cut with Spe1 and Pst1. Ligated the pieces together; tried both an insert:backbone ratio of 6:1 and 3:1.

Tuesday, August 19

Transformed into E. coli to amplify the plasmid.

Wednesday, August 20

Saw no growth on the plates which was inconclusive. Started making new competent cells.

Thursday, August 21 and Friday, August 22

Made competent cells. Retransformed and plated the cells. Incubated overnight.

Saturday, August 23

The control plates had no growth and there were 3-5 colonies on all the other plates. Picked the largest colonies off a 3:1 plate and a 6:1 plate. Made an overnight culture.

Sunday, August 24

Miniprepped the overnight cultures in duplicate.

Tuesday, August 26

Digested miniprep product with EcoR1 and Pst1 to make linear DNA. Ran a gel to determine if plasmid had correctly assembled and transformed. The gel failed.

Thurday, August 28

Using the same transformation we made new overnight cultures for miniprepping at a later date.

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