5' RACE - (Apr/16/2008 )
This is first time we try 5' RACE, and we use other lab's old stuff (Ambion kit).
Although we have 2 bands that can see, but a lot of smear.
We run this to determine the RNA initiation stie, which we don't know how many and how big they will be.
I have no idea did I get it or not.
Can anyone tell me did I have it or not?
Is there any way I can do to reduce smear?
(I have try increase annealing temperature after RT step, PCR and its nest PCR, but it get worse)
If we have it can those 2 bands enough for cloning?
U better do one or two nested PCR with internal primers depending on the availability of known sequence u have. If you are not getting any fragments in nested PCRs mean the primary product u got may be not the one u r looking for.
you can also try touch down PCR whcih may help to avoid any non specific bands and smear.
all the best
Thanks for suggestion
I am current doing gel purification (my boss has no patient to wait for better result) prepare for cloning.
I will use fraction of them for secondary PCR with touchdown protocol.
Hope it will be a good one.