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.

Dig-PCR-Labeling

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 
                                              digoxigenin-11-dUTP)                    
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
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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
Thailand
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