concentration of cDNA after reverse transcription? - (Jun/10/2005 )
How can i measure the concentration of cDNA after reverse transcription?
Thanks!
Ummm well you can't. Thats because the RNA is in there too. I suppose you can take an A260 before and after the rxn and subtract... if you are willing to submit your RNA and cDNA to possible contamination from the cuvette.
We just assume if there was 1ug of RNA in the RNA there is 1ug of cDNA out of the rxn. We know this is incorrect, but use it anyway.
We just assume if there was 1ug of RNA in the RNA there is 1ug of cDNA out of the rxn. We know this is incorrect, but use it anyway.
How about if I need cDNA for creating plasmid. Should i purify the cDNA and how can I measure the concentration of it? Or do I still assume that "1ug of RNA in the RNA there is 1ug of cDNA"?
Okay, I am confused.. why would you need cDNA to create a plasmid... Or are you wanting to clone the cDNA into the plasmid to create a cDNA library.
Okay, the rule of thumb is that 20% of RNA gets converted into cDNA during an rt rxn (dont ask me to remember where I got that number from, it is just rattling around in the back of my mind and I trust it).
So lets make up an rt rxn with 5ug of RNA and assume you get 1 ug of cDNA. How to purify... okay well we have 1 ug of cDNA.. easy enough to visualize on a gel even if it is a smear (assuming you are not using a gsp). Lets be clever and run it all out on a LMP gel... that way we can size select at <500 bp. purify it out of the gel with the 'ol qiagen gel purification kit and then go ahead and clone into the plasmid.
Well wait, what about the cDNA ends.. are they engineered to go into the plasmid... or are you really needing cDNA to make a plasmid and all of this has been a waste of my time because I don't understand why you need cDNA to make a plasmid?
Hi,
Depending up on a vector, the end of cDNA does not have to be "engineered". Over-hanged cDNA surely helps inserting into the vector in correct way, but that's all about it.
Unless you ligate linker arms to the ends of the cDNA, how are you going to ligate it into a plasmid? cDNA does not have sticky ends unless it is engineered into the reverse primer during first-strand synthesis.