Team:NYU Abu Dhabi/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!
  • RPA Specificity test on Bsal DNA

    RPA Specificity test on Bd DNA

Wednesday, 21/10/2020

  • CRISPR Experiments were ran on RPA reactions done on Tuesday, 20/10/2020
  • CRISPR Experiments failed, no fluorescence was observed by the FQ reporters.

Thursday, 22/10/2020

  • Miniprepped Bd 2 and Bsal 4 cultures. DNA concentration was measured using nanodrop.
  • Used Nanodrop to determine concentration of all 8 DNAs
  • Optimized CRISPR protocol and reran the experiment on Bd and Bsal
    • DETECTR Protocol on Bd worked
    • DETECTR Protocol did not function on Bsal.

    CRISPR DETECTR worked on Bd, producing fluorescence

    Troubleshooting required for Bsal CRISPR. Potential issue with sgRNA

Tuesday, 27/10/2020

  • Proof of concept experiment for DETECTR on Bd
  • Successful detection of Bd from RPA to CRISPR/Cas12a to fluorescence by FQ reporter cleavage (DETECTR).
  • DETECTR Proof of Concept for Bd. Ran Triplicates and a negative control.