Hi all,
I am doing reverse transcription for my RNA isolate to proceed to RT-PCR using such cDNA products. we have a non heated lid thermal cycler, so I have to add some mineral oil on my RT mix to avoid the evaporation. After ending the RT, I try to remove the layered oil from my cDNA product, so can do PCR . However, I am still worried if any remnant of the oil may interfere with the reporting of the fluorescence in RT-PCR (signal capture)
Is this possible ?
thanks in advance!!
Why don't you try it without the mineral oil and incubate in a waterbath?
Back in the old days I simply used a waterbath for the reverse transcription and inactivated the reverse transcriptase after the reaction for 5' in a 95°C thermal block. I never had problems with evaporation. People only use cyclers for convenience.
If you don't have a hammer, use a stone ;)
Why don't you try it without the mineral oil and incubate in a waterbath?
Back in the old days I simply used a waterbath for the reverse transcription and inactivated the reverse transcriptase after the reaction for 5' in a 95°C thermal block. I never had problems with evaporation. People only use cyclers for convenience.
If you don't have a hammer, use a stone ;)
There is no need to add mineral oil. The reaction time, volume, temperature in RT reaction will not allow a terrific evaporation.
Dont need to add it in rt-pcr. In thermal cycle the evaporation is minimum. You should close well the eppendorf to avoid stem leakage. If you do realtime pcr also dont need
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