User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/07/12

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Bench work

  1. Miniprep (done with Daniel Catt)
    • Follow instructions for miniprep
    • measure concentration of DNA
    • store @ -20°C
    • Forgot to make glycerol stocks
      • after spinning cells, but before re-suspending them, streaked a plate with samples of each cell pellet.
      • → 37°C ~6h
  1. Glycerol stock cultures (done by Daniel Catt)
    1. 3 mL LB + 3μL 100 mg/mL Amp + V(His)+I colony #1 cells from plate in step 1 above
    2. 3 mL LB + 3μL 100 mg/mL Amp + V(His)+I colony #2 cells from plate in step 1 above
    3. 3 mL LB + 3μL 100 mg/mL Amp + V(His)+I colony #3 cells from plate in step 1 above
    4. 3 mL LB + 3μL 100 mg/mL Amp + V+I colony #1 cells from plate in step 1 above
    5. 3 mL LB + 3μL 100 mg/mL Amp + V+I colony #2 cells from plate in step 1 above
    6. 3 mL LB + 3μL 100 mg/mL Amp + V+I colony #3 cells from plate in step 1 above
    • → 37°C w/ shaking O/N
  2. Sequencing
    • send miniprep samples for DNA sequencing: pTXB1His+BSA colony #1 and pTXB1+BSA colony #2
    • Use T7 promoter and terminator universal primers

Results

  • Miniprep on samples worked without issue:
Results of miniprep: DNA quantitation
Sample (20x dilute) A1 (Abs @ 260nm) A2 (Abs @ 280nm) Ratio A1/A2 [DNA] @20x dilution (μg/mL) [DNA] @1x (μg/mL)
pTXB1+BSA Colony 1 0.263 0.141 1.8644 13.135 262.7
pTXB1+BSA Colony 2 0.638 0.330 1.933 31.9 638
pTXB1+BSA Colony 3 0.541 0.278 1.946 27.05 541
pTXB1+His+BSA Colony 1 0.350 0.183 1.913 17.5 350
pTXB1+His+BSA Colony 2 0.310 0.156 1.987 15.5 310
pTXB1+His+BSA Colony 3 0.397 0.202 1.965 19.85 397
  • Samples sent off for sequencing with T7 and T7 term primers

(7μL of sample + 7μL sterile dH2O):

  1. pTXB1+BSA Colony 1
  2. pTXB1+His+BSA Colony 1
  • Kathryn Muratore 15:27, 19 July 2011 (EDT): Sequencing reactions did not work. pTXB1+BSA with T7 promoter primer had some readable sequence, but a BLAST shows only a short homology with adenovirus.
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