Alice:LabNotes/2010-2-20: Difference between revisions
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==post-hybridization amplification and purification== | ==post-hybridization amplification and purification== | ||
#Prepare the Post-Capture | #Prepare the Post-Capture PCR Master Mix in a 1.5ml tube. | ||
#Aliquot | #Aliquot 5μl of bead-bound captured DNA as template into 3 PCR tubes/wells. Mix well by pipetting up and down 5 times. | ||
The amount of each reagent needed is listed below: | The amount of each reagent needed is listed below: | ||
Phusion High-Fidelity PCR Master Mix (2x) | post-capture DNA: 5ul | ||
PCR grade water | Phusion High-Fidelity PCR Master Mix (2x) 50μl | ||
Syb_FP5, 100 μM (Final Conc.: 2μM) | PCR grade water 41ul | ||
Syb_RP7, 100 μM (Final Conc.: 2μM) | Syb_FP5, 100 μM (Final Conc.: 2μM) 2ul | ||
SYBR green 50x | Syb_RP7, 100 μM (Final Conc.: 2μM) 2ul | ||
Total : | SYBR green 50x 0.4ul | ||
Total : 100ul | |||
98C 30sec -> (98C 10sec -> 60C 30sec -> 72C 30sec) x20 cycles -> 72C 5min -> hold at 4C | 98C 30sec -> (98C 10sec -> 60C 30sec -> 72C 30sec) x20 cycles -> 72C 5min -> hold at 4C | ||
TBE gel image result: | TBE gel image result: | ||
*Qiaquick column purification and elute in 50ul EB | *Qiaquick column purification and elute in 50ul EB |
Revision as of 17:49, 22 February 2010
Agilent SureSelect Human Allexon capture
- previous preparation steps can be found under labnotes 1-15-2010 for CViB
- ligation step and beyond for CViB can be found under labnotes 2-15-10
library hybridization
- Aliquot 16ul (or 779.2ng) of prepped CViB(48.7ng/ul) 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
- 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 1:1 dilution of the RNase Block (purple cap)
- add 1ul 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 (2/22/2010)
- 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.
The amount of each reagent needed is listed below: 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) x20 cycles -> 72C 5min -> hold at 4C TBE gel image result:
- Qiaquick column purification and elute in 50ul EB