Results

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Notebook

To read the detailed protocols of each day, please refer to the protocols to each corresponding day through this pdf file.

Wednesday, 30/09/2020

First day of experiments!

Prepared LB+Ampicilin Agar plates for E.coli cultures.
Prepared Agar Broth
Heat Shock Transformation practice run

Sunday, 04/10/2020

Bd and Bsal gBlock resuspended into solution
PCR Primers 1 and 2 For Bd were resuspended
PCR Primers 3 and 4 for Bsal were resuspended
Ran 4 Q5 High Fidelity PCR Reactions on Bd and Bsal with primer sets 1-4

Monday, 05/10/2020

Ligated each PCR Products onto 4 separate pJET vectors
Transformed our pJET vector+PCR products into competent E.coli through heat shock. Competent E.coli was spread across separate agar plates.

Tuesday, 06/10/2020

Inoculated E.coli containing Bd and Bsal genes

Transformed Product with Bacteria Colonies before Inculation

Wednesday, 07/10/2020

Cultures that were inoculated on Tuesday, 06/10/2020 were miniprepped.
Respective DNA concentrations were measured using a nanodrop
Miniprepped DNA was used to run a PCR reaction as a quality control for Thursday, 08/10/2020

Thursday, 08/10/2020

Gels were prepared for DNA Gel electrophoresis
Gels were ran on Bd and Bsal DNA from all primer sets

Bsal Gel ran perfectly
Bd Gel had issues with the PCR reaction ran on Wednesday, 07/10/2020.

UV Bsal Gel Quality Control 8/10/2020

UV Bd Gel Quality Control 8/10/2020.png

Sunday, 11/10/2020

Bd DNA from the miniprep prepared on Wednesday, 07/10/2020 was used again to run a PCR reaction
More E.coli that had Bsal were inoculated.

Bsal 4 Agar Plate

Bd 2 Agar Plate

Monday, 12/10/2020

E.coli that was inoculated on Sunday, 11/10/2020 were miniprepped to have larger stock of Bsal DNA. The DNA concentrations were measured using a nanodrop
PCR reaction from Sunday, 11/10/2020 was ran on a gel.
PCR reaction failed, once again. Suspected that the primers stock was contaminated.

Bd Miniprep PCR 12/10/2020

Tuesday, 13/10/2020

New Bd primers were resuspended and were used to run another PCR reaction on Bd
RPA reactions were ran on Bsal
Reaction results were observed on a gel.

RPA reaction on Bsal worked!
Bd PCR finally worked on primer set 2. We can proceed with amplification on Bd

Bsal RPA Gel Results 13/10/2020

Bd Miniprep PCR Gel 13/10/2020

Wednesday, 14/10/2020

8 RPA reactions were ran on Bsal to check for specificity
More E.coli containing Bd DNA was innoculated

Thursday, 15/10/2020

Miniprepped Bd 2 cultures that were inoculated on Wednesday, 14/10/2020. The DNA concentrations were measured through a nanodrop

Monday, 19/10/2020

RPA reactions were ran on Bd to check for specificity
Results were not ideal. Bands were too faint for Bd and two out of three RPA reactions for Bsal failed. Have to re-try tomorrow

RPA Specificity test on Bd DNA

RPA Specificity test on Bsal DNA

Tuesday, 20/10/2020

8 RPA reactions for both Bd and Bsal each were ran to check for specificity
Gels were ran for all RPA reactions
Gel results were perfect! RPA for Bd and Bsal are specific to their own primers!