As an example, I suppose to keep the cycle 1 in 22 C for 5 mins while I kept it for 5 seconds.....42 C for 30 minutes while i only kept it for 3 seconds. I guess when I was entering the numbers in the machine, totally forgot the basic training steps. Should I throw away my samples and start again ?
Hello
Yes, I would throw them away and don't think abut them anymore! If you have other samples, better to make cDNA for all of them in the same way and correctly.
it probably depends a bit on whether expression is high in your gene of interest, It is not quite as bad as you think because primer binding and extension happen at all temperatures around the set temperature while the cycler is getting down to temp and then rising and extension will be happening at room temperature while the sample is sitting around at the end of the reaction so if the expected cdna is quite short I would pcr amplify it and see if it has worked
- The short answer is YES those abbreviated times will affect your RT reaction
- If that were me I would not use the samples you describe
- Your new times are much more sensible
- For more efficient random priming by th way (I assume you are priming with random hemamers from your 22C temp) I would follow that reaction by a 4C for 5 minutes (o stabilise primer binding) before going into your 42C RT step
- In fact, if this were me, I would program following:
- 1. 22C 10min
- 2. 5C 5 min
- 3. Pause
- 4. Bring out and place at room temp for about 10min
- 5. Put tubes back and commence a second program cycle starting with:
- 6. 42C for 1 hour (longer RT times contrary to expressed wisdom do improve RT efficiency)
- 7. 85C 5 min (to kill RT and melt duplexes)
- 8. 5min 4c
- 9. Cold soak (4c) until you are available to stop PCR program and remove tubes
- Badges
- Science method