Hi, here in my lab I have a kit for RT-PCR with probes (for the real time quantification). Can I run my reaction without adding the probes, in order to obtain en “end point” product? I was thinking about running two reactions, one without the probes and one with the probes, in order to obtain the product amplified but also its quantification.
I want to do this because I have to amplify a gene from a ssRNA+ virus and insert it into a plasmid.
Do you think it is possible? Does the absence of the probes impacts negatively on the Taqman polymerase? Thank you for your help
You don't need an extra reaction, you can just take the samples at the end of the qPCR reaction as your endpoint PCR product.
However, in qPCR you generally only amplify a small part of the transcript so you will probably need to set up a separate reaction with a different primer set in order to get the full gene for cloning.
Hello,
The first reaction you need to do is RT, which will allow you to obtain DNA from RNA. This is done in a water bath at 37°C for 1 hour, using reverse transcriptase.
Once this reaction is completed, you move on to qPCR, but this time, for quantification, you must design primers that will amplify just part of your obtained DNA and not all of it (you must test before effectiveness of your primers).
@Yoav Lubelsky but if i designed my primers in order to amplify only my target gene (after the retro transcription), it should not be partially amplified, or am I wrong?
Do you use oligo-dT or random primers for the RT step?
With oligo-dT you should theoretically get the cDNA for to complete transcript, you can than design PCR primers to amplify the entire thing or just the CDS in order to clone it. Another option will be to set a different RT reaction with a gene specific primer in order to get the cDNA just for your gene for the purpose of cloning.
@Yoav Lubelsky yes, I will use oligos for the RT. I also have designed primers to the CDS that I need
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