User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/08/24

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Objective

Repeat BSA cloning into intein vectors with new, correct 3' primer.

Bench work

  1. PCR purification
    • Followed instructions for Promega Gel and PCR purification kit, using spin column method
    • Purified experimental BSA PCR sample from yesterday
  2. Double-digest
Tube sterile H2O 10X NEB2 100X BSA DNA NheI (10 kU/mL) SapI (2 kU/mL
pTXB1His 14.5 μL 5 μL 0.5 μL 25 μL of 102 μg/mL 2.5μL 2.5μL
pTXB1 14.5 μL 5 μL 0.5 μL 25 μL of 120 μg/mL 2.5μL 2.5μL
BSA PCR 0 μL 5 μL 0.5 μL 39.5 μL from purified BSA PCR reaction in step 1 2.5μL 2.5μL
  • →2h @ 37°C
  1. Gel purification
    • Kathryn Muratore 17:28, 24 August 2011 (EDT):I forgot to phosphatase the vectors before loading the gel, so we will phosphatase after purification.
    1. 50μL pTXB1His double-digest from step 2 + 10μL 6X loading buffer
    2. 10μL 1KB ladder
    3. skip
    4. 50μL pTXB1 double-digest from step 2 + 10μL 6X loading buffer
    5. 50μL BSA PCR double-digest from step 2 + 10μL 6X loading buffer
  2. Spun down 4 x 500mL growths from yesterday (10 minutes at 3810rpm and 4°C)
    • Resuspended pMAL-pIII in 100mL 30mM TrisCl 20% sucrose w/v pH 8.0 then placed in freezer @ -20°C°C
    • Resuspended pMXB10 in 25 mL 20mM TrisCl 500mM NaCl pH 8.5 then quick froze with liquid nitrogen → freezer @ -80°C
  3. After gel purification, added 6μL 10x phosphatase buffer, 3μL dH2O, and 1μL phosphatase to the 50mL the two plasmids were eluted into.
    • Heat inactivated phosphatase with 5 minutes at 65°C
Tube sterile H2O 10X ligase buffer BSA plasmid T4 DNA ligase
pTXB1 + BSA 13μL 5μL 20μL 10μL 2μL
pTXB1 (-) 33μL 5μL 0μL 10μL 2μL
pTXB1.His + BSA 13μL 5μL 20μL 10μL 2μL
pTXB1.His (-) 33μL 5μL 0μL 10μL 2μL

Results

  • GEL


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