Post-DD problem

Maaria Roschier mroschie at jalus.uku.fi
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
---
Maaria Roschier (mroschie at jalus.uku.fi)
Department of Neuroscience and Neurology, University of Kuopio, Finland.






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