RT-PCR - a lot of wrong size fragments. - (Jan/28/2005 )
Hello dear colleagues.
I'm sure, this question might look stupid, but one have to forgive me since I'm novice in the RT-PCR.
So, the question is:
I'm trying to RT-PCR two genes. One of them (sure, it is GAPDH) is looking nice, but the other is just a number of fragments or a smear across the entier length of a lane ( ).
What I tried:
Changed the temperature for the right primer annealing.
Changed the temperature of PCR.
Ordered the second pair of primers and used them in different combinations with different temperatures.
Nothing good happens.
lower Tm until you get distinct bands. haven't really done RT-PCR though, so this is from a normal PCR point of view.
If your mRNA is less abundant mRNA, try Touch-down PCR .