https://www.neb.com/tools-and-resources/troubleshooting.../pcr-troubleshooting-guid

hi

did you test your primers by using one silico PCR (just to see if the primers amplify the right sequence and have an idea on Tm..). try primer-blast from the NCBI website to ensure you're right (https://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome). the followings steps are at bench, but you didn't precise the conditions.

fred

Hello Fiorella,

Why did you divide the gene in 3 fragments? Would not it be easier to amplify a single 4kb fragment?

I would suggest, if the gene is problematic and you think it can have similar sequences in the genome, that you do nested PCR: you design primers that amplify a bit more than the whole gene, then you do the PCR, dilute (1:200 is normal) it, and use that as template for a second PCR with primers that amplify a shorter fragment that spans the whole gene (look up "nested PCR" for an explanation.

The polymerase is very important for long genes, so I would suggest you use one like Phusion or Q5, or similar, with proof-reading activity.

Once you clone the whole fragment in a plasmid, you can design primers on it to have it Sanger-sequenced.

Best,

Ruben

You can start with rising annealing temperature as much as possible (the highest tempereture for which your PCR is still yielding a product). The second option is to  design so called nested PCR.

I tried to amplified in a single fragment but it was not possible, just one accession we could amplified with a single sequence. From these sequence we design new primers but it doesn't work, the first fragment that includes one exon is very difficult amplified. Also I tried different annealing temperature but the maximum is 50C degrees and the minimum is 40C (which is too low), when we increase the temperature there is no band. And when we get a specific band with the correct size, we cloned but then  we read the sequence and results that s not the correct fragment. I tried nested PCR but the band is too week, and it works with the minimum temperature. 

Hi florella

how do you design the primers? what you can also do is to test touchdown PCR to increase the possibility to amplify something. since it's the first exon with probably the promotor and substantial common set sequences, this point could be at the origin of your false sequence results. can you blast the sequence and see in it where you could design more specific primers?

fred