User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/06/07

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AU CHEM-570 Lab Prep Main project page
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Objective

  • Create a new empty vector that has an intein, chitin binding domain, and his-tag.

Bench work

  1. QuikChange
    • template = 10 μg/mL pTXB1
    • forward primer = 12.5 ng/μL ins-His-after-CBD
    • reverse primer = 12.5 ng/μL Rins-His-after-CBD
    • anneal step = 7 min
    • 18 melt/anneal/extend cycles
    • Kathryn Muratore 12:07, 9 June 2011 (EDT): I had the wrong volume of 10X buffer in the original protocol. Therefore, these reactions were run with 2x buffer, not 1x buffer.
  2. analytical minigel
    1. 10 μL 1KB ladder
    2. --
    3. 5 μL pTXB1 QuikChange experimental reaction
      • Lost some sample while loading
    4. --
    5. 5 μL pTXB1 QuikChange control reaction
    6. -10 --
    • → 90V ~1h
    • stain in EtBr and rinse in TAE
  3. DpnI digestion
    • add 1 μL DpnI to experimental and control tubes from step 1
    • → 1h @ 37°C
    • → store at 4°C

Results

Image:Gel_dna_110607_annotated.jpg

  • Expect ~6.7 kb band in lane 3
  • Hard to tell here, but there may be a very faint band at the right size in lane 3.
  • Nothing was loaded in the last lane, but we confirmed that the "band" and "smear" are in/on the gel. This can not currently be explained.
  • CONCLUSION: QuikChange failed. Will have to repeat.
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