Post-DD problem

Maaria Roschier mroschie at
Thu Jan 8 08:15:54 EST 1998

Hi netters!

We have been checking our cloned DD bands using colony PCR combined with
reverse slot-blot hybridization. The problem is that the method works
well for longer (1 - 1.5 kbp) PCR products but poorly for short (200 -
400 bp) products for which only a faint signal is seen. The protocol is
summarized below.

First, we perform colony-PCR of pBluescript inserts using T3 and T7
primers.  (The products look OK  in agarose gel.) Next we denature the
PCR samples with 0.4 M NaOH (10 min / 95 C), then vacuum transfer them
using a Bio-Rad slot-blot apparatus onto Hybond N+ membrane, followed by
an additional denaturation with 0.4 M NaOH.

We prepare our probe by reverse transcribing total RNA to cDNA in the
presence of 32P-dCTP. The resulting probe has an approximate size range
of 500 - 1500 bases with a label incorporation of 50 - 60 %. The
(pre)hybridization conditions are: 6x SSC, 5x Denhardt's, 0.5% SDS, 0.1
mg/ml salmon sperm DNA, 68 C o/n.

Any suggestions to increase hybridization sensitivity towards shorter
targets would be welcome!

Thank you,
Maaria Roschier (mroschie at
Department of Neuroscience and Neurology, University of Kuopio, Finland.

More information about the Methods mailing list