Shear gDNA (Hydroshear, Gene Machines) speed code 4, 30 shearing cycles, wash 3x, 2x, 5x with acid, base, TE washes as recommend by the manufacturer. (Digesting with a random four cutter is an option but may cleave in the oligo sequence for a significant percentage of the oligos. The target size is between 1-2kb. Shearing too much will result in a lower labeling efficiency as the klenow will have less room to bind on very small pieces of DNA.)
Take 2ug of DNA and bring the volume up to 23µl with ddH20.
Add DNA to 2.5X Buffer/random octamer mix (15µl random octamer at 1µg/µl + 5µl 10X NEBbuffer2 per reaction).
BOIL 5min to denature, ice.
On ice add 5µl 10X amino-allyl dNTP mix (final concentration of reaction mix should be 3mM dA, dC, dG, and 1.2mM dT, and 1.8mM of aa-dU).
Add 1µl Klenow enz (USE Concentrated Klenow, 40-50 units per microliter available from NEB). Mix and incubate at 37˚C for 2 hours.
Stop reaction by adding 5µl .5M EDTA pH 8.0
Clean using Amersham Cyscribe post labeling kit:
For every 20-100µl of cDNA reaction place a GFX column in clean collection tube. Add 500µl capture buffer and then add sample. Pipette to mix. Centrifuge at max speed immediately 30s. Discard flow thru. Wash 3x with 600µl of wash Buffer. Centrifuge 1 min to dry completely. Elute in 60µl of.05M NaBicarb run through the column 2X. Dry down in speed vac and resuspend in 30µl ddH20 when ready to use.
Resuspend new tube Cy3 and Cy5 in 24µl DMSO, divide into 1. 5µl aliquots and dry down. Add sample to dry aliquot. Mix and let sit 1 hr in the dark.
Add 4.5µl Hydroxylamine to quench reaction. Mix and incubate 15min RT in the dark.
Combine Cy3 and Cy5 reactions. Repeat clean up with GFX column.
Dry down in speed vac.
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