Alice:LabNotes/2010-2-20

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Revision as of 21:01, 20 February 2010 by >Zsakura2 (→‎library hybridization)
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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)