Alice:LabNotes/2010-2-19: Difference between revisions

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#mix the component according to the chart below at RT to prepare hybridization buffer (if precipitate forms, warm the hybridization buffer at 65C for 5 minutes):
#mix the component according to the chart below at RT to prepare hybridization buffer (if precipitate forms, warm the hybridization buffer at 65C for 5 minutes):
                               1 reaction      4 reaction              5 reaction
                               1 reaction      4 reaction              5 reaction
  SureSelect Hyb #1                25ul                 100ul                   125ul
  SureSelect Hyb #1                25ul               100ul             125ul
  SureSelect Hyb #2 (red cap)        1ul         4ul                  5ul
  SureSelect Hyb #2 (red cap)        1ul                 4ul                  5ul
  SureSelect Hyb #3 (yellow cap)           10ul                40ul              50ul
  SureSelect Hyb #3 (yellow cap)       10ul                40ul              50ul
  SureSelect Hyb #4               3ul              52ul                     65ul
  SureSelect Hyb #4                                     3ul              52ul               65ul
  Total             49ul                196ul                  245ul
  Total                                       49ul                196ul                  245ul
#prepare the SureSelect Oligo Capture Library Mix for target enrichment (keep everything on ice for next few steps):
#prepare the SureSelect Oligo Capture Library Mix for target enrichment (keep everything on ice for next few steps):
##add 5ul of SureSelect Oligo Capture library and 1ul ddH2O
##add 5ul of SureSelect Oligo Capture library and 1ul ddH2O

Revision as of 19:15, 19 February 2010

Agilent SureSelect Human Allexon capture

Overview and time requirements

File:Agilent sureselect protocol.jpg

Step Time
gDNA library production 1 day
library hybridization 24-72 hrs
bead preparation 30 minutes
capture selection and washing 2 hours
DNA purification 30 minutes
Post-hybridization amplification 1 hour
PCR purification 30 minutes
size selection and purification 4 hours

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

library hybridization

  1. Aliquot 500ng of preped gDNA libraries and concentrate at <45C down to 3.4ul. If the samples dried up completely, resuspend in 3.4ul ddH2O.
  2. mix the component according to the chart below at RT to prepare hybridization buffer (if precipitate forms, warm the hybridization buffer at 65C for 5 minutes):
                             1 reaction      4 reaction              5 reaction
SureSelect Hyb #1                 25ul               100ul              125ul
SureSelect Hyb #2 (red cap)        1ul                  4ul                  5ul
SureSelect Hyb #3 (yellow cap)        10ul                40ul               50ul
SureSelect Hyb #4                                     3ul               52ul                65ul
Total                                       49ul                 196ul                  245ul
  1. prepare the SureSelect Oligo Capture Library Mix for target enrichment (keep everything on ice for next few steps):
    1. add 5ul of SureSelect Oligo Capture library and 1ul ddH2O
    2. prepare 1:1 dilution of the RNase Block (purple cap)
    3. add 1ul of diluted RNase Block to each capture library and mix thoroughly by pipetting
    4. keep tubes on ice
  2. prepare library for target enrichment in a separate tube (for the following steps, make sure evaporation is no more than 3-4ul):
    1. put 3.4ul of prepped library(500ng) into tube B add 2.5ul of SureSelect Block #1, 2.5ul of SureSelect Block #2, and 0.6ul of SureSelect Block #3
    2. mix well by pipetting and cap the tubes
    3. heat up the prepped libraries + blockers at 95C 5min and 65C hold
    4. maintain the tubes at 65C while load 40ul of hybridization buffer into tube A (# of tubes needed is the # of prepped libraries)
    5. incubate both tubes at 65C for at least 5 min before go on
  3. put the SureSelect Oligo Capture Library from step 3.4 into tube C and incubate at 2 min (now the thermocycler should have three tubes per sample incubating at 65C)
  4. while maintaining the tubes at 65C, transfer 13ul from tube A (hybridization buffer) into tube C (SureSelect capture library) for each sample
  5. while maintaining the tubes at 65C, transfer the entire content from tube B (prepped libraries) into tube C (SureSelect capture library) for each sample
  6. mix well by pipetting and the hybridization mix should be around 27-29ul
  7. incubate the hybridization mixture at 65C for 24-72 hrs with heated lid at 105C (make sure no extensive evaporation if incubating more than 24hr)

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.