Alice:LabNotes/2011-1-29: Difference between revisions

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=Agilent SureSelect Mouse Exon capture=
*Previous library construction step using Nextera kit can be found under labnote 1-28-2011


==library hybridization==
#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.
#mix the component according to below at RT to prepare hybridization buffer (if precipitate forms, warm the hybridization buffer at 65C for 5 minutes):
##For every reaction, add 25ul of SureSelect Hyb #1, 1ul of SureSelect Hyb #2 (red cap), 10ul of SureSelect Hyb #3 (yellow cap), 13ul of SureSelect Hyb #4, total is 49ul
##For every 4 reactions, add 100ul of SureSelect Hyb #1, 4ul of SureSelect Hyb #2 (red cap), 40ul of SureSelect Hyb #3 (yellow cap), 52ul of SureSelect Hyb #4, total is 196ul           
#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
##prepare 1:3 dilution of the RNase Block (purple cap), combine 1ul RNase Block with 2ul ddH2O
##add 2ul of diluted RNase Block to each capture library and mix thoroughly by pipetting
##keep tubes on ice
#prepare library for target enrichment in a separate tube (for the following steps, make sure evaporation is no more than 3-4ul):
##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
##mix well by pipetting and cap the tubes
##heat up the prepped libraries + blockers at 95C 5min and 65C hold
##maintain the tubes at 65C while load 40ul of hybridization buffer into tube A (# of tubes needed is the # of prepped libraries)
##incubate both tubes at 65C for at least 5 min before go on
#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)
#while maintaining the tubes at 65C, transfer 13ul from tube A (hybridization buffer) into tube C (SureSelect capture library) for each sample
#while maintaining the tubes at 65C, transfer the entire content from tube B (prepped libraries) into tube C (SureSelect capture library) for each sample
#mix well by pipetting and the hybridization mix should be around 27-29ul
#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)
==Prepare magnetic beads==
#prewarm SureSelect Wash Buffer #2 at 65C for use in next step "Select hybrid capture with SureSelect"
#resuspend and vortex the Dynal magnetic beads (Dynabeads M-280 Strapatavidin, invitrogen)
#for each hybridization, add 50ul dynalbead to 1.5ml tube
#wash the beads:
##add 200ul of SureSelect Binding Buffer
##mix the beads on a vortex for 5 sec
##put the tubes into a magnetic separator, remove and discard the supernatant
##repeat the above steps (4.1-4.3) for a total of 3 washes
#resuspend the beads in 200ul of SureSelect Binding buffer
==Select Hybrid capture with SureSelect==
#estimate the volume of hybridization mix that remained after 24-72hr incubation
#add hybridization mix directly from the thermal cycler to the bead solution, and invert the tube to mix 3-5 times
#incubate the hybrid-capture/bead solution on a nutator or shaker at RT for 30min (make sure the sample is properly mixed)
#briefly spin in a centrifuge
#separate the beads and buffer on a magnetic separator and remove the supernatant
#Resuspend the beads in 500ul of SureSelect Wash buffer #1 by mixing on a vortex for 5 sec
#incubate the samples for 15 min at RT
#wash the beads:
##separate the beads and buffer on magnetic separator and remove the supernatant
##resuspend the beads in 500ul of prewarmed SureSelect Wash Buffer #2 and mix on vortex for 5 sec
##incubate the samples for 10min at 65C
##invert the tube to mix as the beads may be settled
##repeat the above steps (8.1-8.4) for a total of 3 washes, and make sure all the wash buffer has been removed
#mix the beads in 50ul of SureSelect Elution buffer on a vortex mixer for 5 sec to resuspend the beads
#incubate the samples for 10 min at RT
#separate the beads and buffer on Dynal magnetic separator
#transfer the supernatant to a new 1.5ml tube and add 50ul SureSelect Neutralization buffer to captured DNA
#Desalt the capture solution with Qiagen Minelute column and elute in 15ul EB
==post-hybridization amplification and purification==
*Prepare the Post-Capture PCR Master Mix in a 1.5ml tube.
*Aliquot 5μl of bead-bound captured DNA as template into 3 PCR tubes/wells. Mix well by pipetting up and down 5 times.
                                                          x3
post-capture DNA:                              5ul
Phusion High-Fidelity PCR Master Mix (2x):    50μl
PCR grade water:                              41ul
Syb_FP5, 100 μM (Final Conc.: 2μM):            2ul
Syb_RP7, 100 μM (Final Conc.: 2μM):            2ul
SYBR green 50x:                              0.4ul
Total :                                        100ul
98C 30sec -> (98C 10sec -> 60C 30sec -> 72C 30sec) x 13 cycles -> 72C 5min -> hold at 4C
*Qiaquick column purification and elute in 40ul EB

Latest revision as of 23:33, 7 February 2011