Real time PCR supermix - (Jun/01/2005 )

Hello,
Has anybody ever tried to make RT-PCR supermix by their own and has it worked?

Please HELP!!
Thanks

What exactly is the problem?

Prior to being able to purchase Supermixes (late 1990s-early 2000s), you pretty much had to make your own. Therefore, yes, I have made and produced successful RT-PCR data from "home-made" supermixes i.e. combining the 5-10x buffer, MgCl, dNTPs, water, primers and polymerase.

Note: if you want to make your own, do not add the polymerase until just prior to usage. Many enzymes have endonuclease activity and will chew up your primers. Some are also sensitive to the buffer conditions.

Hope this helps,

Sasha.

Dear Aussie,
Are you talking about PCR supermix or RT-PCR supermix?
You seems never mantion about RTase and Hotstart Taq.

Do you have any idea about a buffer system which can function for both RT and Taq enzyme?

Thank you very much

Regards
Hadrian

Hello

I am sorry, I am new to this forum so I actually have not figured as to how to use so this might be the wrong place for my msg!!!!


After quite a few trials I got a decent regression value, yet the slope is very poor about 1.2. what could have gone wrong?
It would be great if any of you have any suggestions!!!

b

Dear Abi,
Yes : ) I think you are right. You have been to the wrong forum room : )
We are talking about RT-PCR. How about you?

Regards
Hadrian