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=Biotin-labeled probe capture continued= *previous steps can be found under labnote 2/15/10 *use only the DF and foreskin sets to do the following steps to find the ideal condition ==Prepare the Streptavidin Dynalbeads== #Take 50ul M-280 streptavidin Dynalbeads per reaction, place the tube on magnet and remove the liquid when the solution becomes clear #add twice the volume of the beads of 1x binding buffer, vortex for 5 sec, place the tube on magnet and remove the liquid when the solution becomes clear #wash the beads for a total of 3 times with the 1x binding buffer (1M NaCl, 10mM Tris-HCl pH 7.5, 1mM EDTA) #resuspend the beads in 200ul 1x binding buffer, warm up to 45C. #Add the 20ul hybridization mix to 200ul M-280 beads, incubate at 45C in the Thermal Mixer at 300rpm for 30 min. #Remove the liquid from the beads with a magnet. Take out 1/2 of the DF set to go on without washing. ==post-hybridization washing== #Perform three 10-min wash with 0.5ml pre-warm 0.1x SSC and 0.1% SDS at 47C (1/2 DF set), 55C, 37C (take 1/2 foreskin set for these temp) using Thermal Mixer. #After the final wash, resuspend the beads with 50ul 0.1M NaOH, incubate at RT for 10min. #Separate the supernatant from the beads with a magnet, and transfer the supernatant (eluted DNA) to 70ul 1M Tris-HCl, pH 7.5. #Purify the 120ul neutralized DNA with a Qiaquick column, eluted with 30ul EB. ==Post-capture PCR== reaction setup: x2 x4 2x Phusion master mix: 50ul 100uM Sol_up 0.5ul 100uM Sol_lo 0.5ul 50X SYBG I 0.4ul Captured DNA 15ul H2O 34ul Perform real-time PCR with 98C 30s -> 20 x (98C 10s -> 63C 20s-> 72C 20s) -> 72C 2min. 14 cycles for DF set without any washing involved *Terminate the reaction when the amplification curves approach to the plateau. *PAGE gel image result: [[File:ZhangLab_2 2010-02-24 11hr 16min.jpg]] *measure enrichment level using QPCR (following the procotol from Nimblgen/Roche)
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