detection of PCR products by hydridization in streptoavidin coated plates

Alejandro Palacios palaman at TERRABA.FING.UCR.AC.CR
Sat Aug 17 10:31:54 EST 1996


Hi neters,

I need help.

I am trying to make an in house PCR detection products system, we are 
interested in a plata hibridization based detction.  I have tryed to 
capture a biotinilated PCR product (only one strand labeled) in 
Sprettoavidin coated plates, after that denaturing with NaOH, 
hibridization with a DIG labeled probe and developing with antiDIG-AP 
with (pNPP).    
The problem is that everything in the tests, including negative controls

I suspect am not doing a good streptoavidin coating or there is too much 
affinity between pyloestherene and the probe. (0.1 microg/well in a 
IMULON 2 strip).

Is any specific way of coating and blocking plates for nucleic acid 
detection?
Is the concentration of streptoavidin too high?
Sugestion of other systems?
All comments welcome.

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Alejandro Palacios         palaman at terraba.fing.ucr.ac.cr
International Center for Medical Research and Training (ICMRT)
Loussiana State University -  Costa Rica
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