Kun:LabNotes/Haplotyping/2010-8-19

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Whole chromosome MDA

  • Objective: in this experiment, I will try to amplify haploid human chromosomes with MDA.
    • Will try on both chromosomes in highly condense form (chr.S) and loosely packed chromosomes (chr.L).
    • Will do protease digestion to disrupt the chromosome structure.
    • Will compare metaphase chromosomes with cell lysates that contain high molecular weight gDNA.
    • Will compare MDA with pWGA.
    • Will also use purified gDNA as control.
  • Number of reactions:
                    1 chr     10 chrs     100 chrs
   chr.S             x 4        x 4         x 2
   chr.L             x 4        x 4         x 2
                    0.6pg       3pg        30pg        NTC
   HMW gDNA          x 4        x 4         x 2
   Pure gDNA         x 2        x 2         x 2         x4

Prepare Alkaline Lysis Solution (ALS) and Neutralization Solution (NS)

  • ALS: (400mM KOH, 100mM DTT, 10mM EDTA)
    • Prepare 5M KOH: weigh one KOH pallet, add nuclease free H2O to make 5M KOH. Make fresh solution every week.
    • Mix 8ul 5M KOH, 2ul 0.5M EDTA, 10ul 1M DTT, 100ul nuclease free H2O.
  • NS buffer (0.4M HCl, 0.6M Tris.HCl pH 7.5):
    • Prepare the 1N HCl Stock solution.
    • Mix 4ml 1N HCl with 6ml 1M Tris.HCl pH 7.5.