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)

  • I'll make these solutions using the DLB and Stop solution from Qiagen's Repli-g kit.
    • Add 500ul RT-PCR grade water to the DLB tube.
    • Add 10ul DLB solution into 70ul RT-PCR grade water. Split into 4 tube (20ul/tube).
    • Add 8ul Stop solution into 72ul RT-PCR grade water. Split into 4 tubes (20ul/tube).
    • Perform UV treatment on the solutions for 5 minutes.

Whole cell lysates

  • Yesterday I collected ~3 millions GM20431 cells (442,000/ml x 6.8ml), and lysed them with 200ul Cell Lysis Buffer (20M EDTA, 10mM Tris.HCl , 200mM NaCl, 0.2% Triton X-100, 0.1AU/ml Qiagen Protease).
  • The resulting cell lysate is extremely sticky. I added 800ul EB buffer, still quite sticky. The DNA concentration is roughly 18ug per ml or 18ng/ul.
  • Take 20ul, add 100 UV-treated water -> 3ng/ul;
  • Make 1:10 serial dilution to 300pg/ul, 30pg/ul, 3pg/ul and then 1:5 dilution to 0.6pg/ul. Pipette slowly to avoid breaking HMW DNA.