Post-DD problem

Mart Speek mspeek at tamm.eenet.ee
Fri Jan 9 02:42:39 EST 1998


Maaria Roschier (mroschie at jalus.uku.fi) wrote:

: 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.
(stuff deleted)

Dear Maaria;

The problems you might have could be related to the following:

* DNA/RNA sticks to the filter under high ionic strength contitions
 (mostly provided by 1.5 M NaCl). It is more critical for shorter
  NA fragments (frequently these can pass through-the-membrane during
  traditional and slow capillary transfers), cf, eg, 10xSCC for DNA and
  20xSCC for RNA transfer.
  This is the essence of NA binding to nylon/nc filters. 
  As in your somewhat detailed description you dont mention high salt
  concentration (but 0.4 n NaOH), I assume you are not using them.

* Dropping the size of your PCR products from ~1 kb to 0.2 kb could also
  mean, that hybridization signals could become 5 times weaker. Is'nt
  that true?

* Finally, I am sure you know that, just to remind you that sensitivity
  can be also determined by stringency of washes.

Hope these suggestions will help!

Mart

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