Technical data

4 Hybridization
Step 2. Hybridize gDNA library pools to the SureSelect Capture Library
58 SureSelect
XT2
Target Enrichment System for Illumina
4 In a PCR plat
e (kept on ice), for each hybridization reaction well,
combine the indicated volumes of SureSelect
XT2
Capture Library and
dilute RNase Block, according to Table 26. Mix well by pipetting.
Table 26 Preparation of Capture Library/RNase Block mixture
5 To each well containing 7 µL of Capture Library/RNase Block mix, add
37 µL of SureSelect XT2 Hybridization Buffer. Mix well by pipetting.
If precipitate is present in the Hybridization Buffer, warm the solution to 65°C for 5 minutes
before use.
6 Cap the wells, then briefly spin the plate in a centrifuge or mini-plate
spinner. Keep the plate at room temperature until it is used in step 7.
7 Maintain the gDNA pool plate at 65°C while you use a multi-channel
pipette to transfer the entire 44-µL of Capture Library mixture from
step 5 to each sample well of the gDNA pool plate. Mix well by slowly
pipetting up and down 8 to 10 times.
The hybridization reaction wells now contain approximately 60 µL.
8 Seal the wells with domed strip caps. Make sure that all wells are
completely sealed. Place a compression mat over the PCR plate in the
thermal cycler.
9 Incubate the hybridization mixture for 24 hours at 65°C with a heated
lid at 105°C.
Samples may be hybridized for up to 72 hours, but you must verify that
the extended hybridization does not cause extensive evaporation in the
sample wells.
Capture Library Size Volume of Capture Library per
hybridization reaction
Volume of dilute RNase Block per
hybridization reaction
<3.0 Mb 2 µL 5 µL of 10% solution
>3.0 Mb 5 µL 2 µL of 25% solution
NOTE
CAUTION
Wells must be adequately sealed to minimize evaporation, or your results can be
negatively impacted.
Before you do the first experiment, make sure the plate and capping method are
appropriate for the thermal cycler. Check that no more than 8 µL is lost to evaporation
under the conditions used for hybridization.