Alice:LabNotes/2010-2-19
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Agilent SureSelect Human Allexon capture
Biotin-labeled probe capture continued
- previous steps can be found under labnote 2/14/10
- use only the foreskin set to do the following step first to find the ideal condition
Prepare the Streptavidin Dynabeads
a. Take 50ul M-280 streptavidin Dynabeads 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. d. Remove the liquid from the beads with a magnet. Take out 1/3 of the product to go on without washing. e. Perform three 10-min wash with 0.5ml pre-warm 0.1x SSC and 0.1% SDS at 55C, 37C (take 1/3 for each temp) using Thermal Mixer. 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
a.Set up a 400ul reaction with Phusion High-Fidelity PCR master mix: 2x Phusion master mix: 200ul 100uM PCR_F 2ul 100uM PCR_R 2ul 50X SYBG I 3.2ul Captured DNA 30ul H2O 163ul b.Perform real-time PCR with 98C 30s -> 20 x (98C 10s -> 63C 20s-> 72C 20s) -> 72C 2min. Terminate the reaction when the amplification curves approach to the plateau.
c.Perform PAGE size selection on 250-350bp amplicons for QPCR quantification and Illumina sequencing.