Hi everybody , I need make cDNA from a low concentration of ARN ( lees than 50 ng).I found some MMLV that make cdna from lows ARN concentrations,but all of these use RNA inhibitors and DTT like extra componenets. I know that DTT is a ARNse inhibitor, so my question is, why i must use other enzimatic RNA inhibitor in the reaccion, is really necesary? Can I use more concetration of DTT in a RT mix like RNA inhibitor? Thank you to all!
When working with RNA, especially in a shared lab, doubly so if you're not a pro quite yet - inhibitors are a good idea. As previous comment suggests, if you have absolutely clean everything, all your plastic is virgin, all your glassware is 400C-treated, all your water is DEPC and you have a nice RNAse-free corner in your lab then you may start removing non-essential ingredients.
DTT is not a very good RNAse inhibitor. It's there to keep enzymes happy.
- If you are worried simply leave out. I have done that without adverse impact
- Just make sure all your reagents are molecular grade cf. certified RNAse and DNAse free and preferably fresh ( so that no prospect for contamination has occurred)
When working with RNA, especially in a shared lab, doubly so if you're not a pro quite yet - inhibitors are a good idea. As previous comment suggests, if you have absolutely clean everything, all your plastic is virgin, all your glassware is 400C-treated, all your water is DEPC and you have a nice RNAse-free corner in your lab then you may start removing non-essential ingredients.
DTT is not a very good RNAse inhibitor. It's there to keep enzymes happy.
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