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 PCR Master Mix in a 1.5ml tube.
*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.
*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:
  post-capture DNA:            5ul
  post-capture DNA:            5ul

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

  1. 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.
  2. mix the component according to below at RT to prepare hybridization buffer (if precipitate forms, warm the hybridization buffer at 65C for 5 minutes):
    1. 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
  3. 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
  4. 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
  5. 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)
  6. while maintaining the tubes at 65C, transfer 13ul from tube A (hybridization buffer) into tube C (SureSelect capture library) for each sample
  7. while maintaining the tubes at 65C, transfer the entire content from tube B (prepped libraries) into tube C (SureSelect capture library) for each sample
  8. mix well by pipetting and the hybridization mix should be around 27-29ul
  9. 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)

  1. prewarm SureSelect Wash Buffer #2 at 65C for use in next step "Select hybrid capture with SureSelect"
  2. resuspend and vortex the Dynal magnetic beads (Dynabeads M-280 Strapatavidin, invitrogen)
  3. for each hybridization, add 50ul dynalbead to 1.5ml tube
  4. wash the beads:
    1. add 200ul of SureSelect Binding Buffer
    2. mix the beads on a vortex for 5 sec
    3. put the tubes into a magnetic separator, remove and discard the supernatant
    4. repeat the above steps (4.1-4.3) for a total of 3 washes
  5. resuspend the beads in 200ul of SureSelect Binding buffer

Select Hybrid capture with SureSelect

  1. estimate the volume of hybridization mix that remained after 24-72hr incubation
  2. add hybridization mix directly from the thermal cycler to the bead solution, and invert the tube to mix 3-5 times
  3. incubate the hybrid-capture/bead solution on a nutator or shaker at RT for 30min (make sure the sample is properly mixed)
  4. briefly spin in a centrifuge
  5. separate the beads and buffer on a magnetic separator and remove the supernatant
  6. Resuspend the beads in 500ul of SureSelect Wash buffer #1 by mixing on a vortex for 5 sec
  7. incubate the samples for 15 min at RT
  8. wash the beads:
    1. separate the beads and buffer on magnetic separator and remove the supernatant
    2. resuspend the beads in 500ul of prewarmed SureSelect Wash Buffer #2 and mix on vortex for 5 sec
    3. incubate the samples for 10min at 65C
    4. invert the tube to mix as the beads may be settled
    5. repeat the above steps (8.1-8.4) for a total of 3 washes, and make sure all the wash buffer has been removed
  9. mix the beads in 50ul of SureSelect Elution buffer on a vortex mixer for 5 sec to resuspend the beads
  10. incubate the samples for 10 min at RT
  11. separate the beads and buffer on Dynal magnetic separator
  12. transfer the supernatant to a new 1.5ml tube and add 50ul SureSelect Neutralization buffer to captured DNA
  13. 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