SEPTEMBER
Monday, September 1
Miniprepped the overnight cultures and analyzed with nanodrop to find the concentrations.
Tuesday, September 2
Talked to our advisor Dr. Peebles about why the part had not assembled correctly. Discussed that our primers for FadD should have Xba1, Spe1 and Pst1 cut sites.
Ran a restriction enzyme digest with EcoR1 and Pst1 to remove the insert and make the plasmid linearized. Also cut the lac promoter plasmid with EcoR1 and Pst1 for the same reason. Ran a gel to determine if the plasmids were correctly assembled. The gel failed.
Wednesday, September 3
Discussed primers and gels with our advisor Dr. Peebles. Discovered that our primers should have a Xba1 cut site not an Xho1 site. Redesigned and reordered primers. Also discovered that the ladder we were using for gels was optimized for TBE not TAE. Got a new ladder that works in TAE.
Thursday, September 4
Choose new lac promoter, part BBa_J04500, from 2014 Distribution Kit, plate 3 well 19J. Transformed part into E. coli.
Friday, September 5
Made culture tubes of and plated lac promoter transformation.
Saturday, September 6
Minipreped the lac promoter. Took concentrations of the miniprep using a nanodrop. Ran a restriction enzyme digest on the lac promoter using Xba1 and Pst1. Ran a gel to check the lac promoter, the gel failed.
Sunday, September 7
Made new culture tubes so we could miniprep the lac promoter again.
Thursday, September 11
Ran a PCR to extract FadD with the new primers.
Wednesday, September 17
Ran a gel to check the PCR, the gel failed. Ran a nanodrop to find the concentrations.
Thursday, September 18
Transformed lac promoter from 2014 and 2013 (plate 3 well 20J) Distribution Kits. Retried the gel using SYBR green in the gel not in the well. The gel failed.
Saturday, September 20
Miniprepped both lac promoters.
Monday, September 22
Did a colony PCR using colonies from the lac promoter transformation. Found the concentration of the miniprepped lac promoters. Ran a restriction enzyme digest on the lac promoter plasmid using Xba1 and Pst1 so it was linear for the gel.
Tuesday, September 23
Ran a gel on the PCR product and the digested lac promoter. The gel appeared to fail.
The backbone was visible but the actual lac promoter was not.
Tuesday, September 30
Ran a PCR with protocol and primers from our PI to determine if our PCR enzymes were still good. Ran a gel with this PCR product and the same digested lac promoter. Used Dr. Peebles’ gel viewer and saw that while the lac promoter was correct, the PCR got nothing. We thus determined that our PCR polymerase was likely faulty.
OCTOBER
Wednesday, October 1
Ran a PCR with our PI’s PCR machine and enzymes. Did both a control with her primers and the FadD.
Number of cycles
Temperature (C)
Length of time
1
98
5 min
5 merge these
98
30 sec
5 three
57?
25 sec
5 cells
72
2 min
25 merge these
98
20 sec
25 two cells
72
2 min
1 merge these
72
10 min
1 two cells
4
hold
Friday, October 3
Ran a gel of the PCR products. The first column is the control and the second two are the FadD PCR product.
Saturday, October 6
Digested lac promoter with Spe1 and Pst1. Ligated FadD PCR product and lac promoter digestion to create final plasmid.
Tuesday, October 7
Transformed final plasmid into E. coli.
Wednesday, October 8
Plates with transformation showed no growth.
Thursday, October 9
Retransformed final plasmid.
Friday, October 10
All the plates including the control had no growth. The transformation failed.
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