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Audrey Heathcote
E116C --> GAA,GAG --> UGU, UGC R334C --> CGU--->UGU or CGC-->UGC
Resuspension
Resuspension
R334C was already resuspended
Resuspended E116C to 100uM 6/6/2022
E116C_CFTR_mut_FOR1 -- added 262uL E116C_CFTR_mut_FOR1 -- added 281uL
E116C
E116C
Mutagenesis PCR and Dpn1 Digestion - 06/06/2022
Mutagenesis PCR and Dpn1 Digestion - 06/06/2022
Procedure- Site Directed PCR (with Q5 mix) and Dpn1
Diluted the 100uM stock solutions of both FOR and REV mutagenesis primers to 10uM
9.0 uL MBG H2O
1.0 uL CFTR_pGHE 7.7ng/uL (Freezer C, box 6)
1.25 uL E116C_CFTR_mut_FOR
1.25 uL E116C_CFTR_mut_REV
12.5 uL Q5 2X Master mix (Freezer C, box 12)
Annealed at 65C
Left in freezer A overnight, retrieved at 9:20am 6-7-2022
Dpn1 digest was performed, placed in the incubator 9:30am 6-7-2022
Gel Electrophoresis - 06/08/2022
Gel Electrophoresis - 06/08/2022
Gel was made 6-6-2022 and ran on 6-8-2022. It was stored in fridge A with a kimwipe over it.
PCR 6/6/2022
PCR 6/6/2022
PCR was a failure. I suspect it was due to the annealing temperature. I will repeat the procedure with an annealing temperature of 54C
Mutagenesis PCR - 06/13/2022 (Redo)
Mutagenesis PCR - 06/13/2022 (Redo)
Procedure- Site Directed PCR (with Q5 mix) and Dpn1
9.0 uL MBG H2O
1.0 uL CFTR_pGHE 7.7ng/uL (Freezer C, box 6)
1.25 uL E116C_CFTR_mut_FOR
1.25 uL E116C_CFTR_mut_REV
12.5 uL Q5 2X Master mix (Freezer C, box 12)
Annealed at 54C (2 degrees C lower than our lowest primer's melting point)
Gel Electrophoresis - 06/13/2022
Gel Electrophoresis - 06/13/2022
Ran a gel on PCR product and E116C+R104C Midiprep products.
Gel Electrophoresis - 06/13/2022
Gel Electrophoresis - 06/13/2022
No band was found for the PCR product, indicating that it had failed again. Third time should be a charm, so this time we are trying a different concentration of plasmid DNA: 25ng/uL CFTR_pGHE.
Mutagenesis PCR - 06/14/2022
Mutagenesis PCR - 06/14/2022
Procedure- Site Directed PCR (with Q5 mix) and Dpn1
9.0 uL MBG H2O
1.0 uL CFTR_pGHE 25ng/uL (Freezer C, box 6)
1.25 uL E116C_CFTR_mut_FOR
1.25 uL E116C_CFTR_mut_REV
12.5 uL Q5 2X Master mix (Freezer C, box 12)
Annealed at 54C (2 degrees C lower than our lowest primer's melting point)
Gel Electrophoresis - 06/14/2022
Gel Electrophoresis - 06/14/2022
PCR was successful! We will be using the 25ng/uL CFTR template DNA from now on.
Dpn1 Digestion - 07/13/2022
Dpn1 Digestion - 07/13/2022
Added 1 µL of DpnI (Enzyme Rack, Freezer A) to the remaining PCR product and digest at 37°C overnight. Write DpnI on the side of the tube. If in a rush, you may add 1 µL of FD DpnI and digest at 37°C for two hours.
Circularization and Nanodrop - 07/14/2022
Circularization and Nanodrop - 07/14/2022
Nanodrop concentration: 885.43 ng/uL
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
E116C
E116C
Only one colony... successful but on thin ice. I am going to proceed and see how it goes.
Negative Control
Negative Control
No bacterial growth, indicates a proper sterile procedure.
Single Colony Inoculation - 07/26/2022
Single Colony Inoculation - 07/26/2022
Finished at 4:57 pm, will return between 8-9 am
E116C
E116C
Broth was cloudy, indicating colony growth and successful single colony inoculation
Negative Control
Negative Control
No colony growth, indicating a sterile procedure.
MaxiPrep - 07/27/2022 - 07/28/2022
MaxiPrep - 07/27/2022 - 07/28/2022
Removed from the incubator at 8:17am.
A very nice sized pellet formed, but I wanted to see if more would precipitate, so I added 1/10th total volume of sodium acetate buffer and will let it sit overnight. Finished at 11:00 am 7-27-22
Resuspended on
First pellet from maxiprep on 7/27/2022
Second pellet from ethanol precipitation on 7/28/2022
Nanodrop - 08/09/2022
Nanodrop - 08/09/2022
2515.4 ng/uL
Gel Electrophoresis - 08/10/2022
Gel Electrophoresis - 08/10/2022
Gel Analysis Lane 6
Sequencing - 08/11/2022 , 08/24/2022
Sequencing - 08/11/2022 , 08/24/2022
First failed due to early termination. I think I diluted the primer wrong.
Redoing
R334C
R334C
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