Kun:LabNotes/Haplotyping/2010-8-19: Difference between revisions

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(Created page with '==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)…')
 
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     Pure gDNA        x 2        x 2        x 2        x4
     Pure gDNA        x 2        x 2        x 2        x4
===Prepare Alkaline Lysis Solution (ALS) and Neutralization Solution (NS)===
===Prepare Alkaline Lysis Solution (ALS) and Neutralization Solution (NS)===
*ALS: (400mM KOH, 100mM DTT, 10mM EDTA)
*I'll make these solutions using the DLB and Stop solution from Qiagen's Repli-g kit.
**Prepare 5M KOH: weigh one KOH pallet, add nuclease free H2O to make 5M KOH. Make fresh solution every week.
**Add 500ul RT-PCR grade water to the DLB tube.
**Mix 8ul 5M KOH, 2ul 0.5M EDTA, 10ul 1M DTT, 100ul nuclease free H2O.
**Add 10ul DLB solution into 70ul RT-PCR grade water. Split into 4 tube (20ul/tube).
*NS buffer (0.4M HCl, 0.6M Tris.HCl pH 7.5):
**Add 8ul Stop solution into 72ul RT-PCR grade water. Split into 4 tubes (20ul/tube).
**Prepare the 1N HCl Stock solution.
**Perform UV treatment on the solutions for 5 minutes.
**Mix 4ml 1N HCl with 6ml 1M Tris.HCl pH 7.5.
===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.

Revision as of 23:16, 19 August 2010

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.