Kun:LabNotes/Haplotyping/2010-8-19

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Whole chromosome MDA[edit]

  • 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 also use purified gDNA as control.
  • Number of reactions:
                    1 chr      10 chrs     100 chrs
   chr.S            x4(1E-1H) x4(1A-1D)    x 2(5G-H)
   chr.L            x4(2E-2H) x4(2A-2D)    x 2(5E-F)
                    0.6pg       3pg          30pg              NTC
   HMW gDNA                   x4(3E-3H)    x4(3A-3D,5C-5D)
   Pure gDNA        x4(4C-4F)                               x2(4A-4B)

Prepare Alkaline Lysis Solution (ALS) and Neutralization Solution (NS)[edit]

  • 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[edit]

  • 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.

Set up MDA reactions[edit]

  • MDA
    Mix 1ul template with 1ul ALS buffer, RT for 3min
    Add 2ul NS buffer
      
    Add 10ul PNP (Primer-nucleotide-protease) buffer 
      10x RepliPhi Buffer    1.4ul
      1mM N6 primer          1.0ul
      25mM dNTP              0.8ul
      0.05AU/ml Protease     0.5ul
      H2O                    6.5ul
    37C 30min -> 70C 15min -> 4C hold
    Add 6ul ED (Enzyme-Dye) buffer
      10x RepliPhi Buffer      0.6ul
      2X SYBR Green I          1.0ul
      RepliPhi Phi29(100U/ul)  1.0ul
      H2O                      3.4ul
    Incubate at 30C for 4h on Opticon4, take one read every 6 minutes.

Results[edit]

  • The concentration of HMW gDNA seems to be under-estimated by 10-fold. Amplification curves came up faster than those from pure gDNA.
  • The concentration of chrL seems to be slightly over-estimated (~2-fold). Amplicons 2E&2F were likely amplified from a subset of chromosomes.
  • The concentration of chrS seems to be under-estimated (~5-fold). Amplicons 1F/1G were likely amplified from a subset of chromosomes.
 File:2010-08-19-gDNA-control-MDA.pngFile:2010-08-19-HsChr-MDA.png
  • What to do next? Purify 1A/1F/1G/2E/2F with Qiaquick columns, shear and make bar-coded Illumina libraries. Sequence all four libraries in one lane (could be mixed with other compatible libraries).
   Yield:
   1A:  39ng/ul x 40ul = 1.56ug
   1F:  29ng/ul x 40ul = 1.16ug
   1G:  24ng/ul x 40ul = 960ng
   2E:  15ng/ul x 40ul = 600ng
   2F:  28ng/ul x 40ul = 1.12ug

Follow-ups[edit]