Team:NYU Abu Dhabi/Documentation/DOCS 20ee279bfcdc46b09c4fb108851b2757/Biology 93d1eff7b0cd4d6ca8529879e773d615/Lab Plan 77e924c386a94c88b6fc31d680855fa1

Lab Plan

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Lab Plan

@Michelle Anne Hughes @Sakura Grant @Zerina Rahic @Marko Susic @Adrian Villanueva @Yujeong Oh

  • PCR amplification and miniprep
    1. PCR Amplify (4hrs)
      1. Can be left overnight at 4˚C or taken out and stored in the freezer at -20˚C (ideal)
    1. Ligation (15-25 mins — can be stored at -20˚C)
      1. PCR product + PJET
      1. gblock + PJET
    1. Transform bacteria on LB/AMP(1.5-2.5hrs → 24hrs to receive results — stored at 4˚C )

      *note: never do this step on a Thursday

    1. Inoculation (18-24hrs — no break between steps 4 and 5)
    1. Miniprep (plasmid purification) → n of plasmid in eppendorf tubes-50µl (stored at -20˚C)
    1. Nanodrop (measure concentration)
    1. PCR miniprep
    1. Run Gel
  • Test miniprep (x3) [purified DNA]
    1. RPA or LAMP (gels & sybr green)
    1. Specificity (primer set amplification)

      → bd & bsal

      → gel and sybr green

      →negative control: with water instead of DNA

    1. Sensitivity:
      1. Serial dilution

        → Initial [ ] = 50 ng/ml

      1. RPA/LAMP Gel and sybr green to find sensitivity range
      1. Serial dilution (using sensitivity range)
      1. Gel and sybr green
      1. qPCR from miniprep

        50 → 0.1

  • CRISPR
    1. Miniprep + RPA or LAMP + Cas12a (x3)
    1. Specificity:

      specificity tests

      Propertybd primerbd guidebsal primerbsal guideoutcome
      bd++✓
      bd++X
      bd+?
      bsal++✓
      bsal++X
      bsal+?

      *test mini prep guide using prof. Song's concentrator*

    1. Sensitivity — only with working reporter gene (no gels)
  • Lysis method

    to be done initially on bacteria and test on fungal samples one acquired from Stephane

    → LB Broth

    1. Inoculate broth

      → count cells/measure optical density (OD)*

    1. Test lysis method
    1. Run PCR (x3)
    1. Run RPA (x3) **
    1. Run LAMP (x3) **

    *we don't know what [DNA] is, but know the number of cells (by counting); can do serial dilution to decrease [DNA] concentration of DNA to desired concentration

    **as separate experiments, run RPA and LAMP with CAS12a

Reference Photos

References

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