How big a problem is cDNA sticking to the plastic? - (May/29/2007 )

Hi.

I get nice real time data (healthy versus sick) on the day I make the cDNA.
I dilute the cDNA 10 times (TE beffer) and use that batch the following days.
The batch is kept at 4 degrees.
I make the cDNA in ABgene PCR plates.

However, the following days, I get results, where the two groups are more and more alike.

Could the plastic play a role in this by binding some of the CDNA?
Is low binding DNA plastic a "must"?
Will addition of salmon sperm DNA help and how much?

Thanks

Peider

Ok I guess I didn't address your favorite reason... yes sometimes nucleic acid and protein can adhere to the plastic of some types of tubes, but I really think that your problem is not heat inactivating the RT enzyme and storing the dilutions at 4C. You should also spin down your tubes after storing at 4C. RT, i am pretty sure, has nuclease activity for proofreading and will chew up your sample if you do not heat inactivate it. This is my opinion. I do not think it is the plastic, but if you think that then try the solutions and verify experimentally...

Adding SS DNA to your reaction may or may not have a negative effect (false priming sites, "molecular crowding" etc.) on PCR but if your PCR is not affected this may work because regardless of whether it is plastic binding or nuclease activity having a bunch of extra DNA will help. It will provide a different substrate for the nuclease you are leaving active by not heat inactivating the RT, and it will provide a "coating" so your cDNA doesn't bind the tube while stored at 4C long term, however I just don't understand why not do the heat activation and store your samples properly and save the trouble. It is up to you.

HTH and good luck.

For anything longer than O/N i would store cDNA at -20°C and it is always better to aliquot to avoid unnecessary freeze/thaw cycles. and of course, heat inactivate your RT enzyme 10 min at 70°C.

Thanks for the help.

The reason for not inactivating the RT enzyme is because the Omniscript protocol says that i can do it, but not that I shall do it.
But after all, having problems, I guess I shall :-)

:-)

Peider