I am using Transcriptor first stand cDNA synthesis kit from Roche..........Somehow Transcriptor RT Reaction buffer is missing........can we make it manually. The concentration is given on kit manual.
Laurence Stuart Dawkins-Hall
The KCl provides stable binding between random hexamers and mRNA target. This increases RT efficiency The only thing I would caution is that a basic reaction mix like the above will work in all RT reactions but you might need to tweet molarities; particularly the Mg concentration as some newer RT enzymes require more Mg than original RT formulations Although let me stress that formula will basically work Tweaking molarities merely has an effect on efficiency and not whether RT will occur or not Just to provide some authenticity and context to what I have said when I first started performing RT reactions in the early 90s it was not uncommon to have to make up most buffers manually; including RT buffer
Dear Sansher
in short yes you can:
http://www.protocol-online.org/biology-forums/posts/41856.html
These buffers have critical co factors: in particular mg is required as a co factor for operations of reverse transcriptases and Tris buffer provides a constant and specific pH
See for example the recipe specified above:
- 250mM Tris ph 8.3
- 15mM mgcl(2)
- 375mM KCl
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