RT-PCR - dilution cDNA prior to PCR?
Posted 02 September 2004 - 04:44 AM
I wonder could you answer a basic question for me: Having performed 1st strand synthesis do I have to dilute the cDNA prior to PCR analysis. I designed primers and am using PCR to amplify the product before electrophoresis but I am confused as to whether I need to dilute the cDNA prior to performing the PCR reation. In the promega protocol it says to dilute to 100ul but in others it doesn't mention it.
Posted 02 September 2004 - 08:33 AM
Posted 03 September 2004 - 05:41 PM
I didn't do dilution until I found the protocol coming with the promega kit, then I think diluting RT products is good in two ways: giving you more templates and making detecting subtle expression changes easier.
Posted 09 April 2009 - 07:16 AM
Posted 09 April 2009 - 08:17 AM
I use ~.5 ug RNA for my cDNA synthesis (can use b/w 50ng-5ug i think)
I use a First Strand Kit (superarray biosciences) and after the reaction I dilute the 20ul reaction into 91ul H20 (as it tells you to do in the protocol)
If the reaction is 100% efficient, which it's most likely not, this will give me a concentration of 45ng/ul
For my reaction I use 15ul template cDNA (~675ng if reaction is 100% efficient)
I use 18s rRNA as my reference gene and it is relatively abundant (~8 Ct value @ 675 ng), but my lower expressed genes show up too (~30 Ct)