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PCR products in Southerns

David Micklem WCI drm21 at mole.bio.cam.ac.uk
Wed Jul 27 17:13:07 EST 1994


In article <316ch7$gm9 at server.st.usm.edu> sywang at whale.st.usm.edu (Shiao Y. Wang) writes:
>Judy Broom (judy at sanger.otago.ac.nz) wrote:
>
>: I would like to use PCR products as probes in Southerns (using 
>: the Boehringer Random Primed DNA Labeling Kit).  What purification 
>: of the PCR product is necessary?  Someone recommended the Bio Rad Pr
>: ep-A-Gel system to me - would a crude ethanol precipitation do in
>: stead?  (I tried probing with NO purification, and it sort of worked... )
>
>Why don't you just make your probe by PCR directly? Gibco/BRL sells a kit
>for doing so but the components are those used for normal PCR. That way you
>can skip the purification step altogether. I'm trying this but am having
>difficulty getting good incorporation. Will post a success story soon I hope.
>
Yes! I do this all the time now, and find it works better than random priming.
The protocol I have (which I got from Michael Akam's lab) is very simple,
 and certainly doesn't require any form of kit!:
 
1) Do a standard PCR of the bit you want to label. I usually only do 20 cycles.
2) If the band you get is reasonably clean, use 0.5ul in a second PCR reaction
  WITHOUT gel purifying first.  Cold nucleotides remaining from the initial PCR
  dilute the hot label, giving a more stable probe. (See 3) if you want a
screaming hot probe which blows itself to bits v quickly).
   THis labelling PCR is done in 40ul with 4ul(or whatever) of a stock of
   2mM each dA,dG,dTTP replacing your normal 2mM dNTP mix, and 4ul of 32P dCTP
(2-3000Ci/mMole, about 40uCi).  After 10-15 cycles, incorporation should be
   about 50-60%.

3) If your band isn't clean enough, or you need an incandescent probe, then
  gel purify by your favorite method first.  THe probe wont all be full length
  but will be 5-10* hotter than by 2).  
  Use about 1% of the DNA from the first reaction in a 20ul PCR mix.  Use 1ul
  of the dAdGdTTP mix (ie 0.5*normal conc) instead of dNTPs, and 4ul 32PdCTP
  as above.  Five cycles should be sufficient, but I don't really ever use this
 method as I find that 2) above works great for everything I've tried. (eg.(
 screening genomic libraries for homologous sequences).

That my 50pence worth - hope it works for you....

David

_____________________________________________________________
D.R.Micklem,
Wellcome/CRC Institute,       Time flies like an arrow...
Tennis Court Road,              
Cambridge CB2 1QR             Fruit flies like a banana.
UK                             
Tel: [+44] (0)223 334129      Email:drm21 at mole.bio.cam.ac.uk
Fax: [+44] (0)223 334089

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