User:Kathryn Muratore/Notebook/AU CHEM-570 lab prep/2011/08/04

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Contents

Objective

  • Run gel on ligations from last night
  • Redo/troubleshoot/transform based on gel + observations

Bench work

  1. Ran DNA gel (1% agarose):
    • Lane 1: Ladder
    • Lane 2: ----
    • Lane 3: pTXB1 double digest 08/02 COULD NOT LOAD
    • Lane 4: pTXB1 double digest; ligated with 'Old' buffer 08/03
    • Lane 5: pTXB1 double digest; ligated with 'New' buffer 08/03
    • Lane 6: ----
    • Lane 7: pKK223-3 BamHI digest 08/03 COULD NOT LOAD
    • Lane 8: pKK223-3 BamHI digest; ligated with 'Old' buffer 08/03
    • Lane 9: pKK223-3 BamHI digest; ligated with 'New' buffer 08/03
    • Lane 10: ----
    • Lane 11: pMXB10 QuikChange +His 08/03
    • Lane 12: pMXB10 QuikChange (-)control 08/03
    • rest blank
  2. Based on an inability to load the digested, cleaned samples, these were re-cleaned with more extensive drying steps in order to remove supposed excess alcohol. The alcohol (2-propanol) would keep the gel load samples from resting in lanes and also inhibit ligase activity.
  3. Re-ligate the test ligation reactions done yesterday with re-cleaned plasmids.
  4. 'Overloaded' ligation of pTXB1.His and pTXB1 from 7/27 (50μL total volume)
    • 20μL ddBSA insert + 10μL pTXB1 + 5μL ligase buffer ('New') + 13μL dH2O + 2μL T4 DNA ligase
    • 20μL ddBSA insert + 10μL pTXB1.His + 5μL ligase buffer + 13μL dH2O + 2μL T4 DNA ligase
    • 0μL insert + 10μL pTXB1 + 5μL ligase buffer + 33μL dH2O + 2μL T4 DNA ligase
    • 0μL insert + 10μL pTXB1.His + 5μL ligase buffer + 33μL dH2O + 2μL T4 DNA ligase

Results

Gel

Image: analytical_8.04.2011labeled.jpg

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