Yes Veteran, it doesn't work, i have tried supplementing it with DMSO up to 5% and with Betaine, but it doesn't work. I am planning to try with ethylene glycol and 1,2-propanediol.
The problem is that both sequences (as i want to keep them below 1kb) are really hard to design primers for, as they are extremely GC rich in most areas, so I am pretty sure the primers I have designed are just the best possible for these specific regions (they do not form any dimers, loops, etc.). I might try increasing the annealing temperature even more and playing dropping the Mg concentration, just to see what happens. Thank you both!
Considering your comments, I guess the best thing to do in my case would be to add RNAse to both lysis buffer in the beginning and to the TE buffer in the end, then measure with nanodrop. In this case, hopefully, i will not have high amount of RNA to interfere with the PCR reaction (whatever the reason for that may be) and will be able to amplify my DNA sequence. Then, i can just run the PCR product on a gel and purify it for sequencing. I guess in this way i should get minimum RNA contamination (with a bit of practice maybe). By the way, they told me to be careful when purifying the amplicon from the gel, as silica or resin might interfere with sequencing as well. Have you heard anything like that?
I was also thinking of RNAse treatment, so i think i will probably go with it. However, the presence of Proteinase K in the buffer would also degrade the RNAse, wouldn't it? So in this case, should i add it in the TE buffer in the end? Do you know if i need to incubate with RNAse for specific period of time to make sure i degrade all RNA, or does it act in the matter of seconds?
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