Kun:LabNotes/Haplotyping/2010-8-19
Jump to navigation
Jump to search
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.