Alice:LabNotes/2010-2-20
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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
- 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)