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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 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)=== *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. ===Set up MDA reactions=== *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.
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