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Leah Chaney, Audrey Heathcote
L218C
L218C
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
Both L218C reactions were successful.
Circularization and Ligation - 07/20/2022
Circularization and Ligation - 07/20/2022
Did not do a gel to confirm, proceeded to transformation.
Nanodrop Concentration
L218C -1 :
L218C -2 :
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
L218C-1
L218C-1
Only two colonies formed, so it was successful but barely. I will be continuing with this plate.
L218C-2
L218C-2
Unsuccessful in the opposite direction: excessive colony growth. I'm not sure why because I did the same 40ng/uL dilution on this DNA and also used 20 uL JM109 cells. I'm going to try diluting further to 20ng/uL and still use 20uL JM109.
Negative Control
Negative Control
No bacterial growth, indicates a proper sterile procedure.
Transformation - 07/22/2022
Transformation - 07/22/2022
Procedure Redo L218C-2
Diluted circularized product to 20 ng/uL, used 1uL DNA and 4uL MBG H2O for the first step.
Used 20uL JM109 cells
L218C- 2 Redo
L218C- 2 Redo
There was one small colony, indicating it was successful but just barely. I will use it and continue with 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
L218C-1
L218C-1
Broth was cloudy, indicating colony growth and successful single colony inoculation
L218C-2
L218C-2
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
Both precipitated pellets, but L218C-2's pellet was too small for my taste. I added 1/10th total volume of sodium acetate buffer and let it sit overnight. Finished at 11:00 am 07/27/2022
L218C-1
L218C-1
First pellet formed
L218C-2
L218C-2
First pellet formed
L218C - 1
L218C - 1
Final pellet
L218C - 2
L218C - 2
Final pellet
Nanodrop - 08/09/2022
Nanodrop - 08/09/2022
L218C -1 : 2221.9ng/uL
L218C - 2 : 1783.8ng/uL
Gel Electrophoresis - 08/10/2022
Gel Electrophoresis - 08/10/2022
Gel Analysis Lanes 4, 5
Sequencing - 08/11/2022 , 08/24/2022
Sequencing - 08/11/2022 , 08/24/2022
L218C - 2 was successfully sequenced
L218C - 1 was not successful, I don't know what happened
I332C
I332C
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