Alice:LabNotes/2010-2-19: Difference between revisions
Jump to navigation
Jump to search
>Zsakura2 |
>Zsakura2 |
||
(2 intermediate revisions by the same user not shown) | |||
Line 107: | Line 107: | ||
98C 30sec -> (98C 10sec -> 60C 30sec -> 72C 30sec) x 13 cycles -> 72C 5min -> hold at 4C | 98C 30sec -> (98C 10sec -> 60C 30sec -> 72C 30sec) x 13 cycles -> 72C 5min -> hold at 4C | ||
*Qiaquick column purification and elute in 40ul EB | *Qiaquick column purification and elute in 40ul EB | ||
=Biotin-labeled probe capture continued= | =Biotin-labeled probe capture continued= | ||
*previous steps can be found under labnote 2/ | *previous steps can be found under labnote 2/15/10 | ||
*use only the DF and foreskin sets to do the following steps to find the ideal condition | *use only the DF and foreskin sets to do the following steps to find the ideal condition | ||
Line 143: | Line 138: | ||
14 cycles for DF set without any washing involved | 14 cycles for DF set without any washing involved | ||
*Terminate the reaction when the amplification curves approach to the plateau. | *Terminate the reaction when the amplification curves approach to the plateau. | ||
*PAGE gel image result: | |||
[[File:ZhangLab_2 2010-02-24 11hr 16min.jpg]] | |||
*measure enrichment level using QPCR (following the procotol from Nimblgen/Roche) | *measure enrichment level using QPCR (following the procotol from Nimblgen/Roche) |
Latest revision as of 21:26, 5 April 2010
Agilent SureSelect Human Allexon capture[edit]
Overview and time requirements[edit]
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[edit]
- Agilent protocol uses Preparing Samples for Paired-End Sequencing (Illumina), our protocol uses NEBNext DNA sample Prep Master Mix Set 1 (NEB, E6040S/L)
- End-Repair reaction (blunt end sample with 3' phos.)
- Qiagen Qiaquick column purification
- Add ‘A’ Bases to the 3' end of the DNA fragments
- Qiagen Qiaquick column purification
- ligate the single-end adapters
- Qiagen Qiaquick column purification (Ampure DNA purification kit can also be used)
- amplify adapter-ligated samples
- 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[edit]
- 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 1:1 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[edit]
- 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[edit]
- 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[edit]
- 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
Biotin-labeled probe capture continued[edit]
- previous steps can be found under labnote 2/15/10
- use only the DF and foreskin sets to do the following steps to find the ideal condition
Prepare the Streptavidin Dynalbeads[edit]
- Take 50ul M-280 streptavidin Dynalbeads per reaction, place the tube on magnet and remove the liquid when the solution becomes clear
- add twice the volume of the beads of 1x binding buffer, vortex for 5 sec, place the tube on magnet and remove the liquid when the solution becomes clear
- wash the beads for a total of 3 times with the 1x binding buffer (1M NaCl, 10mM Tris-HCl pH 7.5, 1mM EDTA)
- resuspend the beads in 200ul 1x binding buffer, warm up to 45C.
- Add the 20ul hybridization mix to 200ul M-280 beads, incubate at 45C in the Thermal Mixer at 300rpm for 30 min.
- Remove the liquid from the beads with a magnet. Take out 1/2 of the DF set to go on without washing.
post-hybridization washing[edit]
- Perform three 10-min wash with 0.5ml pre-warm 0.1x SSC and 0.1% SDS at 47C (1/2 DF set), 55C, 37C (take 1/2 foreskin set for these temp) using Thermal Mixer.
- After the final wash, resuspend the beads with 50ul 0.1M NaOH, incubate at RT for 10min.
- Separate the supernatant from the beads with a magnet, and transfer the supernatant (eluted DNA) to 70ul 1M Tris-HCl, pH 7.5.
- Purify the 120ul neutralized DNA with a Qiaquick column, eluted with 30ul EB.
Post-capture PCR[edit]
reaction setup: x2 x4 2x Phusion master mix: 50ul 100uM Sol_up 0.5ul 100uM Sol_lo 0.5ul 50X SYBG I 0.4ul Captured DNA 15ul H2O 34ul Perform real-time PCR with 98C 30s -> 20 x (98C 10s -> 63C 20s-> 72C 20s) -> 72C 2min. 14 cycles for DF set without any washing involved
- Terminate the reaction when the amplification curves approach to the plateau.
- PAGE gel image result:
File:ZhangLab 2 2010-02-24 11hr 16min.jpg
- measure enrichment level using QPCR (following the procotol from Nimblgen/Roche)