I seek for your advice on the strategy on 5' RACE if I have a sample with cDNA of the patient. As I understood from the literature search, I should start with the 5' priming of the cDNA. In this case, which kit would recommend to choose?
We got good results with the 5'-RACE kit from Roche with at least two half-nested PCR steps.
One caveat: In the end, either sequence the PCR product directly (if you get one clear band) or check at least 6-10 clones if you have to clone it because of multiple bands as there is often some variation in the exact start nucleotide.
I recommend the Race KIT from Clontech (Smarter Race KIT). It works perfect.
@Vasiliy, @Prokash. I have got the remnants of the SMARTer kit from the previous researcher who has left the lab. However, as I understood, in the case of the SMARTer kit one has to start from the mRNA for the 5'RACE experiments, and I only have minute ammount of patient's cDNA, which I have gotten from my colleagues. I read the manual a couple of times, but I can't get how I can proceed with 5-RACE of the cDNA. Could you please help me on this?
I want to know...
is that cDNA is synthesized with gene specific primer ?
what you need is atleast two genespecific primer, terminal transferase, dATP,oligo dt Anchor primer, pcr anchor primer.
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