Can Taq DNA polymerase uses RNA as its substrate? I mixed RT reaction with: dNTPs, RNA of GFP gene, Reverse transcriptase and water and incubated 30 min at 45 °C. Then I mixed PCR with Taq DNA polymerase and as a templat I used 1,5 ul from previous RT reaction. I Saw bands I need but I'm afraid Taq DNA pol could amplify it from residual RNA. I just want to test Reverse transcriptase activity.
Thanks for your responses
Bohuš
No, DNA polymerases don't use RNA as a template. If you want to convince yourself, use the RNA (before RT reaction) as a PCR template.
It sounds like you are concerned if your RT enzyme is working, those kits and enzymes do expire.
Now, it is possible to have DNA carryover into your RNA prep. If your no RT reaction gives a band in PCR it's due to residual DNA (not due to DNA pol using RNA as a template)
Does this help?
@Amy Klocko thanks for your response. I have no band in my no RT reaction. I just ask to make sure myself. So my Reverse transcriptase is active :)
@Amy I produce RT its not RT from the Kit. I used also RT from Kit asi a control
Bohuš Kubala
From QIAGEN:
Can Taq DNA Polymerase use RNA as a template, and generate false positives in "no-RT" controls?
Taq DNA Polymerase has an intrinsic RNA-dependent DNA polymerase activity (reverse transcriptase activity). However, this activity is very low and is only present under buffer conditions that are completely different from those present during PCR. Therefore, "no-RT" controls would not give false positive results due to reverse transcription activity of the Taq polymerase.
https://www.qiagen.com/us/resources/faq?id=65b9b97f-1961-492f-8510-2de502a15159&lang=en
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