I am trying to optimize TMA in my lab. When I start with 150 pg of DNA (Size 235 bp), I get good amplification, which I can visualize as an equivalent size dsDNA product, gel pic attached. However, I feel I don't have any RNA. I am only getting one band corresponding to the dsDNA. Do you think RNA present is getting degraded due to RNaseH activity of RT? temperature profile for TMA is as follows:
94 deg, 2 min for initial DNA denaturation
45 deg, 60 min, Isothermal amplification
85 deg, 5 min
Lanes in the gel:
1. 100 bp ladder, 300 ng
2. TMA product, 235 bp, 1/5th of 20 ul reaction volume
3. Same size PCR product
Thanks
could you try to add DNase to remove the DNA from the sample (and RNA purification), then using RNA loading run the gel for 5min to see whether RNA present?
longer time running gel, the RNA will be lost.
Thanks, Junjie, I tried that too but I could see the remaining little dsDNA while the RNA band was not there.
Hello Sheetal,
Have you checked your T7 promoter oligo sequenced linked to your initial primer that binds to template? Make sure your gel is in denaturing condition made from MOPS diluted in DEPC treated water and formaldehyde.
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