PCR probe

Wasun Chantratita asmsi002 at CMU.CHIANGMAI.AC.TH
Thu May 12 09:04:20 EST 1994

On 11 May 1994, Flavio Solca wrote:

> I am trying to use a 265 bp PCR fragment to screen a lambda gt11 library. 
> Random labeling this probe works reasonably well but the fragments that 
> are produced are a tad small. I hadproblems in getting a signal on my 
> filters.
> Does anyone know a way out?
Dear Solca
	You may try Nonradioactive PCR Labeling techniques. You can 
easily generate either biotinylated DNA probes or digoxigenin labeled DNA 
probes by PCR. You can use lesser template (0.1-1ng) while random 
primer-labeling need at least 25 ng.


1. H2O                 variable
2. 10X PCR Buffer          5 ul
3. MgCl2                1.5-2.5 mM
4. PCR Dig Labeling Mix   5 ul (200 nM dNTP= 2mMdATP,dCTP,dGTPeach, 
                                              1.9 mM dTTP,0.1mM 
5. Primer 1              25 pmol
6. Primer 2              25 pmol
7. Taq DNA polymerase    2.5 unit
8. Template DNA           variable

    Total                50 ul

For Biotin-PCR-Labeling, you may LTI PCR Nonradioactive labeling System.

Both systems work equally well in our hand for detecting DNA targets 
fixed on nylon membrane..

hope this helps

Wasun Chantratita, Ph.D.                        Phone:053-221122 Ext 5086,5068
Department of Clinical Microbiology             Fax:  053-221890 
Faculty of Associated Medical Sciences       Email:asmsi002 at cmu.chiangmai.ac.th
Chiang Mai University
Chiang Mai 50200

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