I have made extraction of brucella DNA from vagnal swab using QIAMP DNA mini kit qiagen then I made real time PCR USING applied biosystem kit for detection brucella sp.
the result is strong positive but after the next day I run that product on gel but
there is no result on gel ?
what is the problem ?
Hi, Israa Hassona,
Real time pcr is based on fluorescence technology to trace the amplification for a specific mutation or genomic contain for specific to virus and bacteria.
It is must that you will have bands, when you run any positive samples in gel, detected by qPCR or Real time pcr.
Note for You :
1. As almost of every assay for Real time pcr is colorless, so when you are going to run on gel, make sure that you have added Loading dye (with the samples) and during gel casting you must need to add Etbr (Ethidium Bromide), as you are analysis the gel unter UV. So Etbr is must.
May be if you find these element okay then you must find bands on gel. Good Luck !
Best regards,
nahid Islam
yes I did all that
loading dye and Etbr
but no result by gel electrophoresis
what is expected causes for that?
Dear Israa,
in my eyes this is question of sensitivity of detection of the PCR products, only. You try to compare apples with pairs!
The sensitivity of detection of the PCR products by fluorescence in RT PCR is at least 100 times higher, as the detection of PCR products with the help of EtBr in gels. Try to make a standard PCR of your positive sample. Perhaps you must use a nested PCR approach.
Best Regards,
Thomas
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