Technical data

Hybridization and Capture 4
Step 1. Hybridize DNA samples to the Capture Library
SureSelect
XT
Target Enrichment System for Illumina Multiplexed Sequencing 61
For each sample library prepared, do one hybridization and capture. Do
not pool samples at this stage.
The hybridization reaction requires 750 ng of prepared DNA in a volume
of 3.4 µL (initial concentration of 221 ng/µL).
1 For prepped libraries with DNA concentrations above 221 ng/µL,
prepare 3.4 µL of a 221 ng/µL dilution of each library.
2 For prepped libraries with DNA concentrations below 221 ng/µL, use a
vacuum concentrator to concentrate the samples at  45°C.
a Add the entire 30-µL volume of prepped library to an Eppendorf
tube. Poke one or more holes in the lid with a narrow gauge needle.
You can also break off the cap, cover with parafilm, and poke a hole
in the parafilm.
b Dehydrate using a vacuum concentrator on low heat (less than 45°C).
c Reconstitute with nuclease-free water to a final concentration of
221 ng/µL. Pipette up and down along the sides of the tube for
optimal recovery.
d Mix well on a vortex mixer and spin in a centrifuge for 1 minute.
3 Transfer each 3.4-µL gDNA library sample (750 ng) to a separate well
of a 96-well plate or strip tube. Seal the wells and keep on ice.
CAUTION
You must avoid evaporation from the small volumes of the capture during the 16 or
24 hour incubation.
If you want to use a different combination of thermal cycler, lid temperature, plates or
strips, and sealing method (strip caps or sealing tape), first test the conditions.
Incubate 27 µL of water at 65°C for 24 hours as a test. Include water in each well that
you might use, including those in the center and those on the edges. Check that you do
not get extensive evaporation. Evaporation should not exceed 4 µL.
For a partial list of tested options showing minimal evaporation, refer to “Alternative
Capture Equipment Combinations” on page 97.