Smearing on ategarose gel of real time pcr product??? - (Jun/14/2010 )

HELLOW EVERYONE

HERE ITS DR DANZ....
I M WORKING ON EXPRESSION STUDY OF TLR GENE IN MASTITIS USING REAL TIME PCR
I AM WORKING ON AB7500 FAST REAL TIME SYSTEM AND GETIING GOOD EXPRESSION-
GOOD AMPLIFICATION PLOTS AND MELT CURVE.

BUT THE PROBLEM IS THAT ,THE PCR PRODUCTS WHICH I GET AFTER RUNNING REAL TIME PCR ARE NOT PROPERLY VISUALISED ON AGAROSE GEL (2%)

THE SAME PRODUCT WHEN DONE BY NORMAL PCR SHOWS BANDC OF APROPRIATE SIZE(200 BP)

I M USING QUIAGEN QUANTIFAST SYBR GREEN MASTER MIX

ANY SUGGESSION IS APRECIABLE

THANKS

PLEASE REPLY

Hello,

Could your qPCR buffer be affecting the gel? There may be something in the qPCR reaction mix that makes running the gel problematic. Check the protocol and instructions which came with your qPCR reaction, there might be some clues there. Perhaps you could try cleaning your PCR product? This might help?