User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/07/20

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Objective

  • Re-quantify plasmid+insert minipreps from previous week
  • BamH1 digest of above-mentioned samples.....
  • Run gel with BamH1 digested samples


Bench work

  1. Analytical digest
    1. 7μL pTXB1 + 1μL 10X BSA + 1μL 10X NEB3 + 1μL BamHI
    2. 7μL pTXB1+BSA#1 + 1μL 10X BSA + 1μL 10X NEB3 + 1μL BamHI
    3. 7μL pTXB1+BSA#2 + 1μL 10X BSA + 1μL 10X NEB3 + 1μL BamHI
    4. 7μL pTXB1+BSA#3 + 1μL 10X BSA + 1μL 10X NEB3 + 1μL BamHI
    5. 7μL pTXB1His + 1μL 10X BSA + 1μL 10X NEB3 + 1μL BamHI
    6. 7μL pTXB1His+BSA#1 + 1μL 10X BSA + 1μL 10X NEB3 + 1μL BamHI
    7. 7μL pTXB1His+BSA#1 + 1μL 10X BSA + 1μL 10X NEB3 + 1μL BamHI
    8. 7μL pTXB1His+BSA#1 + 1μL 10X BSA + 1μL 10X NEB3 + 1μL BamHI
    • 1h 20' @ 37°C
  2. Gel (with agarose at 0.8%)
    • mix 2μL loading buffer with each sample from step 1 above and load on gel in same order, with 1KB ladder in the middle
  3. Double-checked miniprep [DNA] (User:Daniel Catt)
  4. Transformed ligation product from 7/7/11 into competent DH10B made on 7/15/11 using standard transformation protocol (User:Daniel Catt)

Results

Analytical gel

DNA gel 110720 annotated.jpg

  • All miniprep samples are the wrong size. This implies that the ligation failed entirely.

DNA concentrations

  • New [DNA] on miniprep samples from 7/12/11

(Though DNA.....