Here is my story:
1. I used total RNA which I extracted from cell as template, and used AccessQuick RT-PCR Kit (Promega A1702) to do 1-step-RT-PCR. Then I ran agarose gel and got target band.
2. I used total RNA as templated and did reverse transcription with RT Kit (Appliedbiosystems 4368813), and then did PCR with PCR kit (ThermoScientific K1082). After agarose, I got either no band or the band I don't expect.
The target band I want is 1500bp.
I believe RNA and cDNA and primers are okay because 1) I ran agarose gel for RNA before next step. RNA is intact. 2) I used the cDNA in other reactions and got desired bands (from 300bp to 1200bp). 3) Primers are newly synthesized and they work in other reactions.
I feel very sad can't figure out the reason why the results are different. Has anyone met similar issue before?
Thank you guys!
Dear Yao,
Different factors involved with each method (1/and or 2-step-RT-PCR) may produce differing results. Please, check the attached file!!
Kind Regards.
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