2 replies to this topic

[jacobripley]

Hello,

I am quite new to PCR but there is something I don’t understand

Why is it that when I use a No RT ( a sample that hasn’t gone through reverse transcription) as a control I see no amplification - I am aware this is a good thing. But basically why doesn’t the mRNA act as a template and produce no product? after all I have primers and polymerase in there that would recognise it? Is it to do with the U bases preventing primer annealing? But this cannot just be the only thing preventing this amplification. Once the mRNA is turned to cDNA everything appears to be fine.

Thanks
J

[leelee]

Your polymerase is a DNA polymerase, and can therefore only catalyse the polymerisation of DNA and not RNA.

[Trof]

leelee, on Mar 14 2010, 10:35 AM, said:

Your polymerase is a DNA polymerase, and can therefore only catalyse the polymerisation of DNA and not RNA.

To be precise, it can only catalyse polymerisation of DNA with DNA as a template. It doesn't recognise RNA.