DTT & RNase inhibitor in RT reaction - (Nov/28/2005 )
hi! I have been trying out reverse transcription - pcr with varying results. Can someone tell me about his / her experience about the same, while not using DTT and RNase inhibitor? I am specially interested in knowing as to how important DTT is. Also, another problem I am facing is that the cDNA remains trapped in the gel loading well, while the ladder moves on. Any suggestions?
How did you obtain your cDNA? I find if there is any precipitate in the sample it can cause it to stick in the wells.
Regarding the role of DTT in RT reaction, check this thread http://www.protocol-online.org/forums/inde...?showtopic=4770
DTT is usually included in the RT buffer which has a strong smell of DTT.
I think RNase inhibitor is necessary in RT reaction, although I have not tried to leave it out.
I always use an rnase inhibitor, but a colleague of mine is now getting the same results as I am without them... The use of an rnase inhibitor is a must if you're not sure about possible rnase contamination. Most RT-buffers indeed have DTT in there, why would you try without it?
hi! I have been trying out reverse transcription - pcr with varying results. Can someone tell me about his / her experience about the same, while not using DTT and RNase inhibitor? I am specially interested in knowing as to how important DTT is. Also, another problem I am facing is that the cDNA remains trapped in the gel loading well, while the ladder moves on. Any suggestions?I have used single step RT-PCR kit supplied by Finnzymes.
Unles you have high yields of RNA, there is no way that RT will work without inhibitor.
How much RNA are you dealing with?
Why don't you buy RNase inhibitor and DTT? If you can afford reverse transcriptase, why not buy the other two, which are much cheaper?
-Matt
How much RNA are you dealing with?
Why don't you buy RNase inhibitor and DTT? If you can afford reverse transcriptase, why not buy the other two, which are much cheaper?
-Matt
I agree, I thought the reason you have to have RNAse inhibitor is because it is nearly impossible to isolate the RT enzyme with no RNAse contamination and to inactivate one is to inactivate the other so all the enzymes have some level of RNAse contamination?? Maybe this is an old idea and now there are some kind of RNAse free strains or cell-free systems in use for making these enzymes??