Hello,
Previously, I asked a question about cDNA samples.
I am conducting many RNA works so handling cDNA sample is
very important task for me, like any other researchers!
From total cDNA sample, I extract nominated volume of cDNAs according to
my protocol and sometimes I discover that I extract less cDNAs by pipette.
In this case, I think there are two possible options.
1. Discard already extracted cDNAs and extract new cDNAs from total sample.
2. Just pour extracted cDNAs again into total sample again and extract again
almost immediately,
confirming whether right volume is extracted.
In two options above, the 'cDNAs' include diluted cDNAs in real-time pcr,
not only general synthesized cDNAs.
It seems that I prefer second option but I wonder whether it is my choice to
choose between two options above or I must follow specific option between the two or another one I didn't describe above to get reliable result.
Thank you!
Sorry but I didn't understand your question. You performed your RT reaction and after that you are trying to extract your cDNA? Why?
I didn't understand your question either. Extract from where? The cDNA solution from your RT reaction can be used (and should) directly as template for PCR ( you could also do dilutions if you have too much RNA to begin with)
Agustina is right. You should use your RT reaction directly (as long as you don't use a large volume).
Dear Agustina and Andre Fernandes,
Thank you for your support.
It seems that protocols differ between researchers.
I synthesize 20ul of cDNA each sample. and I extract 1ul of cDNA from it
to mix with 19ul pcr mixture and got good results so far but anyway I want to
get confirmed about my question above.
I believe you are mixing the terms.
The term "extract" means you add phenol:chlorophorm vortex and centrifugue the mixture and collect the aqueous phase. This procedure is supposed to remove proteins and other impurities from nucleic acids.
I believe you are saying you "take" 1uL from your cDNA (RT reaction) and simply add to your PCR mix.
Thank you Andre Fernandes,
I am sorry I am just junior in research so I was confused about the terms.
so can I ask about my qeustion now? I think you now understand what I want to ask.
Thank you very much!
If I understood you right you have a pipetting problem. For qPCR your pipettes must be calibrated correctly. You should always start with your large volumes (in this case you PCR mix) and ALWAYS use the appropriate pipette to collect the desired volume.
For exemple:
19uL from your PCR mix you must use a P20 pipette
1uL from your cDNA you must use a P2 pipette
There is always an error during pipetting, so your 20uL cDNA won't allow you to make 20 reactions (you'll probably get 19 reactions and a little left over).
oh i see, yeah you must use a p2,5 to take 1ul of cDNA. Always use a pipette with the higher limit close to the volume you have to take.
For example, 19ul should be taken with a p20 (it goes from 2 to 20), not a p200 (it goes from 20 to 200).
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