really stupid question... - (Sep/27/2006 )
.... this is going to sound stupid coming from someone who has been doing PCR for almost 10 years, but how many of you out there use unrefined RT product as your cDNA template, and how many of you purify the product of your reverse transcription before using a set amount in your real-time PCR reaction???
I use unrefined. after the RT incubation, I put the cDNA at 4C and use it up within a week or so, or store it at -20 for longer term
you can do one-step RT-PCR, so you shouldn't have to purify it. of course, you are using small amounts of template...I suppose if you needed large amounts you'd have to purify it to prevent inhibition from the RT components?
I've always used unpurified first strand cDNA products. Normally there is so little cDNA there that it's very tricky to purifiy it properly and without losing half of it in the process.
what about diluting it... i purchased some pre-validated primers this month and they didn't work [annoying], i spoke to the suppliers and the only thing i hadn't done was diluted or purified and quantified my cDNA, i did that and voila... they worked [kind of]
I use unrefined cDNA with pre-validated primer sets, and it works quite well. I always dilute my cDNA so that I have 10ng/reaction. I have found that I can create an efficiency curve with a dynamic range of 0.01 to 100 ng cDNA, but even the highest concentration requires dilution of my cDNA.
you know what, i've been doing real-time pcr for 2 years with unrefined and neat cDNA, i've been to seminars held by biorad, and read numerous papers on the method and only now have i been told to dilute my cDNA before amplification!!!!
Generally I do a 20ul cDNA synthesis reaction on 1ug total RNA, then I dilute the cDNA 1:5 to 100ul and use 5ul for PCR
this is purely for handling purposes - using 1ul of the cDNA works just as well but you get more consistent pipetting with 5ul (in my hands anyway)
Personally I see no real reason to need to dilute cDNA and I have used the whole 20ul reaction in a single PCR before with nol problems
me either, other than saving on cDNA... all my real-times have worked till now... what a mess.