User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/06/09

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Objective

  • Continue trying to insert the His-tag into pTXB1.

Bench work

  1. QuikChange of pTXB1 with His-tag primers
    1. One reaction (without a corresponding control) following the standard QuikChange protocol
      • compared to last time, the final [buffer] = 1X
      • This reaction went through and extra first 4 steps (see next step below), was pulled out, wax removed, chilled briefly on ice, and 1μL Pfu Turbo added (followed by more wax) before being returned to the PCR machine for the full standard cycling. Thus, the final amount of Pfu = 5U.
      • LABEL: QC pTXB1 2
        QuikChange
        pTXB1+His
        1-step
    2. Another reaction (with corresponding control) following the 2-stage modified QuikChange protocol
      • I accidentally added polymerase to the control tubes (both forward and reverse) at the very beginning
      1. These two tubes were removed and stored @ -20°C after the first cycle.
        • LABELS: ctrl pTXB1 A, ctrl pTXB1 B
          1 cycle
          Pfu added
          QuikChange neg ctrl
          pTXB1+His
          forward, reverse
          2-step
      • I set up new control tubes and put them in the PCR machine for one cycle while the experimental tubes were getting mixed.
      1. I subsequently and mistakenly added polymerase to the mixed control tube when it was added back to the PCR machine
        • LABELS: ctrl pTXB1 A+B
          18 cycles
          Pfu added
          QuikChange neg ctrl
          pTXB1+His
          mixed
          2-step
      2. meanwhile, I mixed the remainder of the negative control reactions, causing them to miss one cycle
        • LABELS: ctrl pTXB1 A+B
          QuikChange neg ctrl
          pTXB1+His
          mixed
          2-step
      • experimental reaction was performed according to the protocol
        • LABELS: QC pTXB1 A, QC pTXB1 B, QC pTXB1 A+B
          QuikChange
          pTXB1+His
          forward, reverse, mixed
          2-step
  2. analytical minigel
    • 1.2% agarose
    1. 1KB ladder
    2. -
    3. Experimental reaction from last time
    4. Control reaction from last time
    5. -
    6. QC pTXB1 2
    7. QC pTXB1 A+B
    8. ctrl pTXB1 A+B
    9. ctrl pTXB1 A+B
    10. -10 --
    • → 1h @ 90V
    • → stain with EtBr
  3. DpnI digestion
    • add 1 μL DpnI to each:
    1. QC pTXB1 2
    2. QC pTXB1 A+B
    3. ctrl pTXB1 A+B
    4. ctrl pTXB1 A+B
    • → 1h @ 37°C
  4. Transform into DH10B
    1. 200 μL Tamra's fresh DH10B + 5 μL 0.2 mg/mL pTXB1 (from original NEB stock)
      • to test competency and also make a stock of this plasmid
    2. 200 μL Older competent DH10B + 5 μL QC pTXB1 2
    3. " + 5 μL QC pTXB1 A+B
    4. " + 5 μL ctrl pTXB1 A+B
    5. " + 5 μL ctrl pTXB1 A+B
    • Follow the standard protocol for all
    • plated on LBAmp
    • → O/N @ 37°C starting around 17:43, 9 June 2011 (EDT)

Results

Image:Gel dna 110609 annotated.jpg‎

  • No bands are apparent at expected 6.7 kb. Appears to have not worked, but we'll see what the transformation results are.
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