DIG DNA labelling kit

New User support at ichr.uwa.edu.au
Tue Mar 21 20:42:23 EST 2000


Could anyone who has ever used digoxigenin to label DNA probes please
provide me with some help? I have recently purchased the DIG High Prime
DNA Labelling & Detection Starter Kit II (Roche) and have been having
problems with checking the labelling effiency. I have been using a
DIG-labelled control DNA (pBR328, linearized with BamH1), as provided in
the kit along side my probe. After applying the control DNA (in serial
dilutions from 0.01pg/ul - 10pg/ul) on a +ve nylon membrane, the DNA was
fixed by crosslinking. It was then blocked, the alkaline phosphatase added
and washed (all buffers provided and diluted according to the
instructions) and incubated in CSPD solution for 5min. The blot was then
exposed to X-ray film for 25min before detection. However, the only
dilution that can be detected was the 10pg/ul spot - the smallest dilution
detectable should be 0.1pg/ul. I have tried crosslinking the membrane at
125mJ, DNA-side up and side down and also at 30mJ. In addition I have also
tried fixing with NaOH. 
Does anyone have any answers?
Thanks
Cheryl

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