I am runing two steps RT-PCR and the target size is 2.2kb. However, my products cannot migrate when I am runing the gel. I measured the concertration of my cDNA using nanodrop and it is around 1300 ng/ul (I know it is not pure cDNA but just a mixture of random primers and dNTP). Why my PCR is not successful? Do I need to dilute my cDNA?
Do you mean to say that you have done a reverse transcription and used the mix for amplifying a 2200bp cDNA( eg. for cloning)? In that case, how much of RNA did you use for RT? If your purpose is cloning, then a working protocol could be to reverse transcribe 500ng-1µg total RNA in a 20-25µl reaction, dilute 10 times or so and use 1-5µl for a PCR reaxn. Your 1300µg/µl looks too much ( I am not sure whether its a typo!). Undiluted cDNA, if used can give a smear (corresponding to the cDNAs) and PCR may not work. Good luck.
Hi Manoj, thank you so much for answering! sorry it is 1300 ng/ul. My orignal RNA sample is around 70 ng in a in a 20ul reaction (I dont have a good sample)
2.2kb is a huge fragment; it will take a long time to migrate through the gel. Following dilution (as suggested by Manoj), use a lower concentration (%) in your agarose gel (if that's what you are using) and run the gel for longer. You will lose your primer bands (at the bottom), but you should see your fragment move out of those wells.
Hi Ashwin, I diluted 5 times and ran the gel for an hour, still got the same thing.
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