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]
- I made three libraries for shotgun sequencing, and the data analysis was done on 09/17.
- Jeff made additional four libraries, and the analysis was done on 12/08.