DYNABEADS - info required

Paulo Magalhaes pamaga at biobase.aau.dk
Wed Oct 6 10:43:28 EST 1993


Hello,

I'm trying some solid phase sequencing for the first time and I've
run into a few difficulties. As I tried to "look" at what was happening
in the different steps, I ran into further difficulties :) So, let's
hear your wise words...

First, what I've done.

1) amplification of the region of interest with normal (non-biotinylated)
   primers -- product = 1.2 kb

   a 1.5% agarose gel shows a nice band -- no smear, or secondary bands

2) re-amplification using ONE biotinylated primer -- yes, I remembered to
   lower the concentration of primers ;)

   at this stage I was still smiling: my agarose gel showed a beautiful
   1.2 kb band, just like the original one

3) cleaning of the "semi-biotinylated" product through Millipore filters
   (to remove any excess primers, Taq, etc, etc)

   so far, so good -- my 1.2 kb sample was still intact

4) immobilization of the PCR product (from 3, above), using Dynabeads --
   of course I washed the beads as stated in the manual! :)

   now the problems began: the "supernatant" removed showed that a *lot*
   of my product had not bound to the dynabeads: the same 1.2 kb band was
   there, although (perhaps) not so intense... a curious note: the BrPhOH
   blue in this sample runs a little slower then other samples (cases 1,
   2, and 3, above, for instance) -- why? (the DNA has the same mobility!)

At this stage I wondered if I had any DNA stuck to the dynabeads -- I ran
a sample of this... the brownish dynabeads were perfectly visible IN THE
WELL, after the run -- are the dynabeads really too big to migrate into the
gel? (1.5% agarose) also, there was no 1.2 kb band visible -- on the other
hand there was a faint "band" in the well (under UV, of course!)

the questions:

a) how can I "see" the *bound PCR product* on a gel? is it possible?
b) why am I loosing so much DNA? (cf. point 4, above)
   yes, I have repeated the procedure -- the results are always like this :)

so, if you have experience in dynabeads and solid phase sequencing, I'll
appreciate your "hints & tips"

many thanks in anticipation

regards from Copenhagen,

Paulo


-- 

**********************************************************************
* Paulo Magalhaes              | email:  pamaga at biobase.aau.dk       *
* Section of Clinical Genetics | fax:    +45 / 31 39 65 43           *
* Rigshospitalet               | voice:  +45 / 35 45 45 92           *
* Copenhagen                   |                                     *
* Denmark                      | snail-mail: C'mon, this is the 90s! *
**********************************************************************
-- 

**********************************************************************
* Paulo Magalhaes              | email:  pamaga at biobase.aau.dk       *
* Section of Clinical Genetics | fax:    +45 / 31 39 65 43           *



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