User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/07/27

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Objective

  • Gel purify double digests from yesterday
  • Transform ligation reactions from yesterday w/ non-ligated counterparts
  • Re-plate transformations from 7/25
  • Start midiprep cultures from streaked plates that grew overnight
  • Perform ligation on double-digested plasmids and insert (from today's samples)

Bench work

  1. Phosphatase reaction with double-digested plasmids from yesterday
    • → added 2μL antarctic phosphatase to the 100μL samples
  2. 2 Gels with the double digested samples:
    • Gel 1: Taped 5 + 3 lanes together for two aliquouts of BSA PCR product → added 100μL to the gel with 1 ladder lane
    • Gel 2: Taped 6 lanes together for each of the plasmid samples → Got 95% of the samples into the gel + 1 ladder lane
  3. Followed protocol for Promega Wizard® SV Gel and PCR Clean-up system for the two gels
  4. Picked 3 colonies from each of the glycerol stock-streaked plates and put into 3mL cultures of LB + Amp for the day
    • Inoculated 100mL cultures at end of day + Amp
  5. Transformed 4 ligation test samples:
    • ("PNL") pTXB1 digested with EcoRI, BamHI, and antarctic phosphatase
    • ("PL") pTXB1 digested with EcoRI, BamHI, and antarctic phosphatase...treated with T4 DNA ligase
    • ("NPNL") pTXB1 digested with EcoRI, BamHI
    • ("NPL") pTXB1 digested with EcoRI, BamHI...treated with T4 DNA ligase

Results

DNA quantitation from gel purification

{{#widget:Google Spreadsheet

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Gels

Gelpurificationplasmids7.27.2011labeled.jpg

Gelpurificationinsert7.27.2011labeled.jpg