User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/08/09

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AU CHEM-570 Lab Prep Main project page
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Objective

  • Run DNA gel with QC product
    • DpnI digest of product (and transformation, if applicable)
  • Re-transform pTXB1 dd + insert samples with greater [DNA]
  • Replate transformations from yesterday (no colonies this morning)
  • Redo QC with pTXB1 control
  • Run gel with serial dilutions of pKK223-3 and another with both cleaned and non-cleaned BamHI-digested pKK223-3

Bench work

  1. DNA gel with QC product from yesterday
    • Lane 1 Ladder
    • Lane 2 pMXB10 @ 10ng/μL (shouldn't actually show up)
    • Lane 3 pMXB10 QC +His
    • Lane 4 pMXB10 QC (-) control
    • Lane 5 ----
    • Lane 6 ~600ng of pKK223-3 miniprep 7/26 by Kay Wang
    • Lane 7 ~300 ng of same sample
    • Lane 8 ~150 ng
    • Lane 9 ~75 ng
    • Lane 10 ~32.5 ng
    • Lane 11 ~16.25 ng
  2. BamHI digest of pKK223-3 miniprep 7/26 by Kay Wang
    • Two samples, identical: 20μL 128ng/μL pKK223-3, 5μL NEB3 10X 5μL BamHI, 0.5μL BSA 100X, 19.5μL dH2O. 37°C for 2 hours.
    • Freeze one digest and clean-up the other with Promega Wizard® DNA clean up kit
  3. DNA gel #2 for the day:
    • Lane 1 Ladder
    • Lane 2 ~256ng of BamHI unclean digest of pKK223-3 above
    • Lane 3 ~128ng of same sample
    • Lane 4 ~64ng
    • Lane 5 ~32ng
    • Lane 6 ~16ng
    • Lane 7 ~8ng
    • Lane 8 ----
    • Lane 9 ~256ng of BamHI cleaned digest of pKK223-3 above
    • Lane 10 ~128ng of same sample
    • Lane 11 ~64ng
    • Lane 12 ~32ng
    • Lane 13 ~16ng
    • Lane 14 ~8ng
  4. QuikChange 2 stage with pMXB10 and +His primers and pTXB1 as (+) control


Results

  • Gel #1:

Image: DNAgel1fromphonesmallersizelabeled.jpg


  • Gel #2:

Image: DNAgel2fromphonesmallersizelabeled.jpg

  • Kathryn Muratore 13:44, 10 August 2011 (EDT): This shows an incomplete digest by BamHI. In Gel #1, you see that the uncut plasmid runs at ~6500 and ~3000 bp. Cut plasmid is expected to run as two bands at 4516 and 268 bp.
    • Top band is uncut plasmid (relaxed)
    • 2nd band is plasmid cut once (4584 bp)
    • 3rd band is plasmid cut twice (4516 bp)
    • 4th band is uncut plasmid (supercoiled)
    • 268 bp piece of DNA is not visible at these load levels and/or was run off the gel.
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