Our laboaratory is genotyping and seuencing candidate genes in different wheat lines, however we are facing often low PCR yield or total failure of PCR of especially resistance gene (R-gene, NBS-LRR) regions. DNA seems fine if test it for a housekeeping gene. Is this a typical case of region specific DNA template degredation or it may be an issue related to region specific DNA methylation.
Your general experiences and tips on PCR success in wheat or barley or specific response to above mentioned questions in particular will be highly appreciated. greetings.
There's plenty of stuff that could go wrong. First of all, the primers. How many pairs could you try for the same gene? Can you get a positive control DNA for testing them and know if they are working? Else you could design new ones with Primer Blast. You could add PCR enhancers. DMSO(1-3%) for example works good on rich GC templates. How long is the product? If its too long, not all TAQ polymerases would work and you may resort to Phusion polymerases. Extension time? Those just at the top of my head.
Give us more details on how are you carrying the procedure.
The fact that your house keeping gene gives you a PCR product would suggest the DNA prep is Ok and methylation shouldn't really affect whether of not you can generate a PCR fragment........so.....to follow on from Esteban.....Are the resistance genes exogenous?? if so have they been inserted as a genomic sequence (to include introns/exons) or from a single ORF? If the former do your primers amplify across an intron exon boundary? You may be trying to amplify a fragment too large for the time of your extension step!
if your housekeeping genes are working then it suggests that the primers for the R gene are degrading. Order new ones and make up in TE not water and make aliquots as primers do not like being thawed too many times.
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