nonradioactive screening

Zainul Zain zainul at KB.USM.MY
Sun Jul 17 23:21:23 EST 1994


We have experience using three types of chemilumisnecent methods: DIG 
(Boehringer), Tropix (can't recall the name of the company) and 
Amersham's ECL. Both the Tropix and DIG systems are far superior to ECL 
(sorry Amersham!). DIG especially gives exceptionally clear background 
and the hybridization buffer containing the probe can be reused at least 
3 to 4 times. If your probe is 200bp in size I am pretty sure you can use 
the random prime method of labelling rather then end-labelling.

We are using the DIG system at the moment in a M. tuberculosis strain 
typing project and have very good results for over 600 different 
isolates. We also use the random prime method to label our probe which 
is around 300bp in size and have also used it to label a 180bp, probe.

Hope this helps.

Zainul

On 17 Jul 1994, Tom Chappell wrote:

> In article <2vuuqa$6bv at nntp2.Stanford.EDU>
> btoomey at leland.Stanford.EDU (Barbara Holland Toomey) writes:
> 
> > I getting ready to screen a cDNA library with a DNA probe. I am interested
> > in nonradioactive methods to label this probe (I have a 200bp PCR product) 
> > since I would like to have kids someday. I am considering Amersham's ECL 
> > 3'-oligolabelling system, but I have no experience with any of the 
> > nonradioactive methods. I would greatly appreciate any advice on this kit or 
> > any others that might be better. Thanks!!!
> > Barbara Toomey
> 
> I've done 230 bp PCR'ed pombe probes onto P1 and cosmid gridded pombe
> libraries with ECL. I get a good positive signal after 2 min exposure
> if probe is labelled with random oligo. Signal is much weaker with FITC
> (?-I think ECL is anti FITC..) incorporation during PCR. I've used it
> as an experiment in the CSH pombe course and it is basically foolproof.
> The only problem I run into is getting enough background to actually
> see the grid pattern on the filters...
> 
> Tom Chappell
> MRC Molecular Cell Biology
> University College London   
> 
> 



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