Embedded Files
Brett Rog, Audrey Heathcote
R334C -- DNA sequence 1166-1168 (1000-1002), CGG
CGG ---> TGC
R104C -- DNA sequence 476-478 (310-312), AGA
AGA --> TGT
Cys: TGT, TGC
R334C
Primer
R104C
Primer
Construct Sequences: https://drive.google.com/drive/u/1/folders/1MjGEGvUCczbZOsxRY70JPuHvORWJ7y8I
Highlighted:
Red- R104C
Purple- R334C
Standard CFTR Protein
Modified CFTR Protein
Resuspending Mutagenesis Primers - 3/22/2022
Resuspending Mutagenesis Primers - 3/22/2022
Spun the primers
Diluted to 100 uMol stock solution for forward and reverse primers
PCR Mutagenesis and Dpn1 Digestion - 3/23/22
PCR Mutagenesis and Dpn1 Digestion - 3/23/22
Tube A: 1uL of 20 ng/uL DNA (dilution of .2 uL of 100 ng/uL DNA), 9.8 uL MBW, 1.25 uL of forward and reverse primers, 12.5 uL Qmix
Tube B: 1 uL of 10 ng/uL DNA (dilution of .1 uL of 100 ng/uL DNA) 9.9 uL MBW, 1.25 uL of forward and reverse primers, 12.5 uL Qmix
Both tubes went into thermocycler with annealing temp of 61 Celsius
After thermocycling over, 2 uL of Tube A and B removed and placed into Tube α and Tube β respectively.
Tube α and Tube β into freezer A, red rack.
1 uL Dpn1 added to remaining contents of both Tube A and B, placed into incubator overnight.
7/18/2022 - Couldn't find it, starting over
PCR Mutagenesis - 07/18/2022
PCR Mutagenesis - 07/18/2022
Site-Directed PCR Mutagenesis with Q5 MasterMix
Used 25uM Template DNA, annealed at 55C
Gel Electrophoresis - 07/19/2022
Gel Electrophoresis - 07/19/2022
Ran for min at 150V for 45 min
R334C-1 was not successful, but I am going to proceed with R334C-2.
Circularization and Ligation - 07/20/2022
Circularization and Ligation - 07/20/2022
Transformation - 07/20/2022
Transformation - 07/20/2022
Diluted circularized product to 40 ng/uL, used 1uL DNA and 4uL MBG H2O for the first step.
Used 20uL JM109 cells I think this is where I went wrong, I didn't use enough cells. It could also be that I didn't need to dilute the 40ng/uL further.
R334C
R334C
Was unsuccessful, no colonies formed. I will try not diluting, just put in 5uL of 40ng/uL and using 40 uL JM109 cells next time.
Negative Control
Negative Control
No bacterial growth, indicates a proper sterile procedure.
Transformation - 07/22/2022
Transformation - 07/22/2022
R334C
R334C
There was excessive bacterial growth probably from too high of a concentration of DNA. There is a small independent colony that I will use for SCI.
Negative Control
Negative Control
No colony growth, indicating a sterile procedure. The date on the plate is incorrect, this negative control was done with 7/22/2022 transformation.
Single Colony Inoculation - 07/26/2022
Single Colony Inoculation - 07/26/2022
Placed in the incubator at 4:57pm, will return tomorrow between 8-9am
Maxiprep - 07/27/2022
Maxiprep - 07/27/2022
Removed from the incubator at 8:17 am
No colony growth. Will redo transformation.
R334C
R334C
Broth was clear, so there was no colony growth, indicating that single colony inoculation was unsuccessful.
Negative Control
Negative Control
No colony growth, indicating a sterile procedure.
Transformation - 08/15/2022
Transformation - 08/15/2022
Redoing, I used 5uL of DNA and 30uL JM109
R334C
R334C
Overgrown bacteria. Something is not right with the DNA, so I'm going to nanodrop again so I can stop wasting bacteria
Negative Control
Negative Control
No bacterial growth, indicating a sterile procedure
Nanodrop showed there was a concentration of 7ng/uL, so my dilution was incorrect. I will be starting over with PCR.
Page updated
Report abuse