I would like to do a PCR reaction of the PITX1 gene and am in the process of designing my primers. I used PRIMER-BLAST but the results only gave me primers that would copy the exons in the gene which means I would have to do 3 PCR reactions for every PITX1 gene I wanted to copy. Would it be possible to copy the whole gene in one go? AccuPrime™ Taq DNA Polymerase claims to be able to do samples 20Kb or less.
Hi there,
if you want to get the full genomic sequence (introns+exons), use the set of primers specific of the 5' and 3' regions of the gene and ignore the ones defined at the exon/intron junctions. And of course if the polymerase is said to be efficient up to 20kb it shoulb be no problem with 6.5kb template.
You could try to hand-design your own primers pair. This is what I usually do, I open my desired sequence through a seqbuilder software, I look at the sequence at 5' for finding a forward primer and also at the 3' sequence for my reverse primer. After finding 3-4 good couple of primers I test the specificity via Primer3 and even via PrimerBLAST and the thermodynamic parameters through oligoanalyzer 3.1.
Bear in mind you're not forced to get a single amplicon for the whole gene, you can design 2 or 3 gene fragments with a slight overlapping region and after the amplification from gDNA you can perform with your purified fragments another overlapping extension PCR and get a single full-length product.
You can use Primer3 (several online interfaces available, e.g. http://bioinfo.ut.ee/primer3/ ) to define in detail your amplicon.
The bigger question here is: What do you want to do with your amplicon? Without that information, it is hard to give you a good answer. What you got from Primer-Blast is most likely the most useful results for sanger sequencing.
1. No need to generate 3 PCRs. If you want to copy the whole gene, you don't have much choose. Make one primer covering the start codon and make another primer covering the stop codon. Adjust the base numbers in the primers so that their Tm will be similar. Then, use a high-fidelity and robust DNA polymerase to amplify your gene. You should be able to amplify the whole unit of your gene if the PCR goes well.
2. From their data (see attachment), it showed that a fat band (6-7 kb) can be amplified with genomic DNA by AccuPrimeTM Taq DNA Polymerase, so your 6.5 kb PCR should be ok, provided you have a good quality gDNA.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Biological Techniques