PCR amplification - how to design the PCR program (Oct/20/2008 )
I have designed the primer using the complete mRNA sequence of the gene along with UTR region(1717bp product size) I tried to amplify with genomic DNA to standardize the PCR program. AT 62 C annealing temp, I got 3 kb Band , i thought it might be due to the presence of introns. then I have proceded to RT-PCR with 62C Anneling temp. but I could not see any band on the gel.
The melting temperatures of both the primers are 57C.
Can anyone help out me with the PCR program.
ThanX a lottttttttttt
Check the Ensembl database or something if there's really an intron and you amplify the right product.
1717bp is quite long for a cDNA, what do you use for RT, random primers or oligo-dTs? Isn't you RNA "shredded" before RT, what method you use to isolate RNA?
Can you guide me how to check for the presence of intron sequences using any good software.
i isolate RNA using Trizol.The gel also seems good for RNA. I use oligo-dts and M-MLV reverse transcriptase for RT.then for second strand synthesis i did PCR with my gene specific primers.then I could only see something shredded at the lower end of the gel.
I would be very thanhful for your suggestion.
Find your gene in Ensembl, then go for the Exon info of your transcript, it will show you exons and introns, find your primers within the sequence and see if there is a intron between them and what length it is.
Your RNA isolation and RT seems fine, if you handle it all carefully and use fresh I don't se any obvious points of trouble. Maybe try to optimise RT-PCR itself, not on DNA level, with some not-as-important samples containing your transcript. We don't usually optimise for DNA when we want to amplify cDNA. If the product is of different length, then you should use a different elongation time anyway.
Thank you TROF
I tried to do as per your suggestion but I think the ENSEMBL seems to be specific for Human and animals genomes databases. I work on plants. how to exon info for plants.
Thanx a lot for your early reply.