PCR- DNA and Tm - (Aug/14/2007 )
I am running real time PCR using the light cylcer on viral cDNA. I was wondering if anyone can tell me how an increase in cDNA concentration can cause a shift in the Tm of the amplicon.
My metling curve looked really strange. the normal melting temp is ~89C and this time there were two abberant peaks almost combining at arounf ~86C.
I normally use sequence specific primers for cDNA conversion and the same primers for the PCR portion
This time i used oligo dt18 primers for the cdna conversion and the usual seq. specific primers for the PCR.
Which primers are better to use for cDNA conversion?
K
-kdavies9-
QUOTE (kdavies9 @ Aug 14 2007, 04:18 PM)
I am running real time PCR using the light cylcer on viral cDNA. I was wondering if anyone can tell me how an increase in cDNA concentration can cause a shift in the Tm of the amplicon.
My metling curve looked really strange. the normal melting temp is ~89C and this time there were two abberant peaks almost combining at arounf ~86C.
I normally use sequence specific primers for cDNA conversion and the same primers for the PCR portion
This time i used oligo dt18 primers for the cdna conversion and the usual seq. specific primers for the PCR.
Which primers are better to use for cDNA conversion?
K
My metling curve looked really strange. the normal melting temp is ~89C and this time there were two abberant peaks almost combining at arounf ~86C.
I normally use sequence specific primers for cDNA conversion and the same primers for the PCR portion
This time i used oligo dt18 primers for the cdna conversion and the usual seq. specific primers for the PCR.
Which primers are better to use for cDNA conversion?
K
You have to be sure you are amplifying the right product. Tm depends on the product, I wouldn't correlate the template cDNA concentration with a shift in the Tm, considering you are actually amplifying what you think you are. Run out the products and sequence them if you can. Tm for a specific sequence should be constant. Extra cDNA is not going to affect the amplicon melting point, but it can affect the efficieny of the reaction.
-Vicky.ac-