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==Biotin-labeled probe capture== *gDNA samples of DF and foreskin will be used, and the preparation protocol can be found under labnote 2-1-2010 ===working buffer preparation=== 1x binding buffer: (1M NaCl, 10mM Tris-HCl pH 7.5, 1mM EDTA) ===Hybrid selection=== Mix the following to 20ul total volume: 1ug ligated DNA with 200ng padlock probes 2.5ug of human Cot-1 DNA 2ul of 10X AmpLigase buffer 1ul of 100uM competing oligos each (Solexa_up and Solexa_lo) [gDNA] DF6-9-9: 221ng/ul (5ul needed) foreskin: 153ng/ul (6.5ul needed) exome probe set 1: w/o PAGE selection (with Jan09 1-5,8,9,Mar R2, R3): 7ng/ul (28ul needed) exome probe set 2: w/ PAGE selection (Jan09 1-5,8,9): 7ng/ul (28ul) system setup (all units are in ul): {| {{table}} | align="center" style="background:#f0f0f0;"|'''reagents''' | align="center" style="background:#f0f0f0;"|'''DF''' | align="center" style="background:#f0f0f0;"|'''DF(for exome probe set2)''' | align="center" style="background:#f0f0f0;"|'''foreskin''' |- | DNA||5||5||6.5 |- | probe||28||28||28 |- | 10x ampligase buffer||5||5||5 |- | human Cot-1 DNA||2.5||2.5||2.5 |- | Solexa_up (100uM)||1||1||1 |- | Solexa_lo (100uM)||1||1||1 |- | ddH2O||6||6||4.5 |- | total:||50||50||50 |- | |} 95C 5min -> cool down to 60C at 0.01C/sec -> 60C 24 hours -> 50C 24 hours. ===Prepare the Streptavidin Dynabeads=== a. Take 50ul M-280 streptavidin Dynalbeads per reaction, place the tube on magnet and remove the liquid when the solution becomes clear b. add twice the volume of the beads of 1x binding buffer, place the tube on magnet and remove the liquid when the solution becomes clear c. wash the beads for a total of 3 times with the 1x binding buffer (1M NaCl, 10mM Tris-HCl pH 7.5, 1mM EDTA) d. resuspend the beads in 200ul 1x binding buffer, warm up to 45C. c. Add the 20ul hybridization mix to 200ul M-280 beads, incubate at 45C in the Thermal Mixer at 300rpm for 30 min, take 1/2 and go on with no wash. d. Remove the liquid from the beads with a magnet. e. Perform three 10-min wash with 0.5ml pre-warm 0.1x SSC and 0.1% SDS at 45C. f. After the final wash, resuspend the beads with 50ul 0.1M NaOH, incubate at RT for 10min. g. Separate the supernatant from the beads with a magnet, and transfer the supernatant (eluted DNA) to 70ul 1M Tris-HCl, pH 7.5. h. Purifythe 120ul neutralized DNA with a Qiaquick column, eluted with 30ul EB. ===Post-capture PCR (done on 3/15/10)=== a.Set up the reaction system with Phusion High-Fidelity PCR master mix: 2x Phusion master mix: 50ul 100uM PCR_F 0.5ul 100uM PCR_R 0.5ul 50X SYBG I 0.8ul Captured DNA 15ul H2O 44ul b.Perform real-time PCR with 98C 30s -> 20 x (98C 10s -> 63C 20s-> 72C 20s) -> 72C 2min. First batch of samples used 20 cycles. For second batch of samples, DF N.W. and DF #2 N.W. only used 15 cycles, while the other had 19 cycles. Terminate the reaction when the amplification curves approach to the plateau. purify the products with Qiaquick columns and elute in 30ul EB Nanodrop result: DF N.W: 177.4ng/ul DF set#2 45C: 34.7ng/ul DF set#2 N.W.: 208.8ng/ul DF 45C: 141ng/ul foreskin 45C: 158.6ng/ul foreskin N.W.: 224.8ng/ul PAGE gel image: [[File:ZhangLab_2 2010-03-15 16hr 33min.jpg]] [[File:ZhangLab_2 2010-03-15 16hr 36min.jpg]]
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