radiolabelling by PCR?

Weining, Song WeininS at prose.dpi.qld.gov.au
Mon Nov 1 04:32:01 EST 1999


I did labelling yearsago with inserts in plasmids. Everything was the same
as normal PCR except cold p32 1/3 of normal concentration, hot p32 the same
as in random labelling.
Song Weining
Leslie Research Centre
13 Holberton Street
PO Box 2282,
Toowoomba, QLD 4350
Australia

Phone: 61-7-46398880
Fax: 61-7-46398800
Email: weinins at dpi.qld.gov.au

From: Zhonglin.Chai at med.monash.edu.au
<mailto:Zhonglin.Chai at med.monash.edu.au> (Zhonglin Chai)
Subject: Re: radiolabelling by PCR?
Date: 25 Oct 1999 16:59:09 -0700
1. You just need to use random priming method by replacing the random
primer with your specific pcr primers, so that you will get a full length
labelled probe DNA. We have used this approach and it works well.
2. Label your DNA ends by kinase (T4 PNK), or kinase label your PCR primers
before pcr.
3. PCR labelling is a bit tricky. It is not reliable in my hands at all.
hope this of help.
zhonglin chai
>HI
>I want to label a PCR fragment of 90 bp length in order to screen a cDNA
>library. As my probe is so short I¥m afraid that random primer labelling
>will be too weak.In Maniartis there are only these random primer
>approaches, nick translation and so on but no radioactive PCR protocols.
>
> Does PCR not work with radioactive nucleotides? Does anybody have a
>protocol for such a labeling PCR? What ratio of labeled an unlabeled
>nucleotide do I have to use? Or has anybody succesfully labeled such short
>fragments by random hexameres?
>
>Thanks!
>
>Gregor




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