Alice:LabNotes/2010-2-19

From ZhangLabWiki
Jump to navigation Jump to search

Agilent SureSelect Human Allexon capture

File:Agilent sureselect protocol.jpg

gDNA sample preparation

  • Agilent protocol uses Preparing Samples for Paired-End Sequencing (Illumina), our protocol uses NEBNext DNA sample Prep Master Mix Set 1 (NEB, E6040S/L)
  1. End-Repair reaction (blunt end sample with 3' phos.)
  2. Qiagen Qiaquick column purification
  3. Add ‘A’ Bases to the 3' end of the DNA fragments
  4. Qiagen Qiaquick column purification
  5. ligate the single-end adapters
  6. Qiagen Qiaquick column purification (Ampure DNA purification kit can also be used)
  7. amplify adapter-ligated samples
  8. Qiagen Qiaquick column purification and PAGE gel verification
  • previous preparation steps can be found under labnotes 1-10-2010 for DF and foreskin sets
  • previous preparation steps can be found under labnotes 1-15-2010 for CViB and CViF sets
  • DF and foreskin sets still have purified post-PCR product after ligation
  • ligation step and beyond for CViB and CViF sets can be found under labnotes 2-15-10

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.