5'end labeling

Zhonglin Chai zchai at COBRA.PATH.MONASH.EDU.AU
Tue Jun 3 22:51:50 EST 1997


>In article <5migiu$o23$1 at sparcserver.lrz-muenchen.de>,
>salger at wap1.zi.biologie.uni-muenchen.de (Klaus Salger) wrote:
>>Halina Sobolewska (hs3 at stir.ac.uk) wrote:
>>: I'm 5'end labeling primers to amplify microsatellites. When I examine the
>>: literature it seems that some say you should use the same molar ratio e.g
>>: 10pmoles of your oligo with 10pmoles of 32P.  One reference labeled
>>: 200pmoles of micro primers with 30pmoles of 32P, another uses a great
>>: excess of the isotope. They were all using 32P with a specific activity
>>: >3000Ci/mmol, 10mCi/ml.
>>: Does anybody have any ideas on this?
>>
>>Halina,
>>
>>if you want to label your primers to a high specific activity you should use
>>an excess of 32P.
>
>Or, you could simply use equimolar (or slight excess of 32P) and incubate
>longer with kinase (like 2 hr).

It is not good to use equimolar (or slight excess of 32P), because the
kinase reaction is in both two directions. If you use equimolar, only half
of the oligo DNA will be labelled when the both directed reactions are
balanced. As a result of hybridization, those unlabelled oligo probes will
hybridize and compete off the hot probes hence reducing the sensitiviy.
Theorectically, you should use 3 to 1 (32P to oligo) molar ratio or higher
to ensure that more than 99% of your oligos will be labelled.



Dr. ZhongLin Chai
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Department of Pathology and Immunology
Monash University Medical School
Alfred Hospital
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