Reamplification of cDNA - (Sep/07/2006 )
We had a precious sample of RNA, we synthesized cDNA, carried out PCR reaction and due to miscommunication are left with a small volume of cDNA sample. We still have to carry out atleast 6 (or we have a way) more PCR reactions, but do not have sufficient cDNA.
DO you have any suggestions??
Can we reamplify the cDNA that we have???
We are in serious trouble...need help.
Any possibility to dilute your cDNA (i.e. is your PCR sensitive enough)?
How does that work???
I think vairus meant can you dilute your cDNA template?
A PCR reaction is an amplification reaction, thus as long as your polymerase is efficient enough, it can came make enough product to produce a detactable signal.
you are currently using 1ul template for a reaction. Can you use 0.4ul (or less, a diluted solution)template for the same reaction instead. if the polymerase is efficient enough, sufficient product will be generate and you will hardly see a difference.
Alternatively, you can do something harder... you could try make more of your cDNA by amplfying it using primers which bind to each end of the cDNA. Like in a conventional PCR reaction.
exacly what perneseblue said.
We for instance do RT-PCR and dertermine detection limits.
We start our series from more than a 100.000 copies or RNA and go down to just several hundreds. So, if we for instance know that we have 50.000 RNA copies and we only have limited stocks left, we know we can use less template than we regularly do, so we can dilute our RNA stock and use it more often.
Indeed, another possibility would be to amplify your entire cDNA and then do your other PCR's like you would do a nested PCR to get the seperate products. I would first try that method on other cDNA to see if you get that first (bigger) PCR working.
How do I design primers for the ends of cDNA? Using smaller amount of cDNA sounds doable, but since i have a very low amount of cDNA i would like to know how to amplify cDNA using primers? I have mutiple primers :- one for 3' and multiple for 5' since I m amplifying Vheavy chain, i am wondering how can I design primers to amplify cDNA?? do you have any specific cDNA reamplification protocols? If so, can you direct me to it?
Just a PCR with primers that span (almost) your entire cDNA. No different from any other pcr you're doing, just longer (therefor you might need some optimalisation, so you better try it on less precious cDNA).