I diluted the DNA templates 10 fold until the smallest one has 1 copy and 5 copies. Then I do q-PCR with following system:
SYBR 5ul
F primer 0.4ul
Rprimer 0.4ul
cDNA 1ul
H2O 3.2
40cycles
The results are strange. The water has an obvious curve similar to 20 copies. The whole process that I did is in the super clean branch.
Can someone tell me how to avoid the negative sample pollution?
Hi Miao
The results your have got is a definitel indications of template contamination in your PCR reagents...........negative sample can be contaminated due to several reasons including 'contamination through pipettes, PCR reagents....so the best way out is to track the source of contamination before proceeding further..
bye
Your ~20 copy Cq amplification (at 35 or 36 cycles probably) is in the typical location for signal being due to primer dimer. As Mickael said - check your melt curve for the primer dimer signature peak. If not primer dimer - then Ajay has spelled out the rest.
Hi Miao,
I also agree with Jack above.....I'll also insist that the PCR amplification of the gene of interest should be well optimized prior to the actual qRT-PCR.....only if you see every thing is working fine you should go for analysis on qPCR instrument...issues related to primer dimers, non-specific bands, should be dealt in the begining.....One should not use qRT-PCR right in the begining by hit and trials....go in a systematic manner...
bye
I do agree with all the above answers....along with melt curve analysis you can simply do electrophoresis of the amplified sample to check whether you obtain an expected band length...that will be supportive to your results...(if it is not the actual amplicon ).
All the best.
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