I used BLAST to check for the specificity of my primers (for RT-qPCR) and could only find the desired product of 67 bp. After analysing with SYBR Green I foundnd two different melting curves for different samples, and gel electrophoresis resulted mainly in a product of ~120 bp (let's call this product A), and in one case the expected ~70 bp (product B). We sequenced the two PCR-products, with poor results. It appears that multiple products are formed and the overall length of the sequenced product was 258 & 560 for product A and 201 & 310 for product B. Oddly enough, only one melting peak per sample is found in the SYBR Green assay. As I mentioned before, this is not found in the BLAST results. Alignment of the sequenced products with the target is impossible due to the poor sequencing results.
Hello Julie Smith ,
In my opinion, your working flow is correct, of course, you need to sequence the products. But, you have to improve the sequencing step.
So, to avoid getting bad results. THE SOLUTION IS: TO CLONE THE PRODUCTS IN A PLASMID and then sequence from the plasmid sequence
pGMT-easy vector is very useful and "easy" to use. This plasmid has the sequences of the T7 and SP6 RNA polymerase promoters. If you use this plasmid remember to use the correct polymerase --> But don´t worry because a lo of polymerases often add a single deoxyadenosine, in a template-independent fashion, to the 3´-ends of the amplified fragments.
Regards! ;)
Félix M.
Sounds like your primers aren't specific and not giving clean melting curves. That should be enough info for you. Try designing a couple other primer pairs using Primer-Blast or another tool. It's probably not particularly interesting to figure out what the non-specific PCR products are.
BLAST isn't 100% perfect. Remember, it's based off what was submitted as a reference genome (usually one individual organism). If the genome is incomplete, then so are the data. It also won't show all the polymorphisms/variations in individuals.
Did you test out your primer annealing temps with regular PCR and a gradient of annealing temps? That could explain some of the issues.
BLAST isn't 100% perfect. Remember, it's based off what was submitted as a reference genome (usually one individual organism). If the genome is incomplete, then so are the data. It also won't show all the polymorphisms/variations in individuals.
Did you test out your primer annealing temps with regular PCR and a gradient of annealing temps? That could explain some of the issues.
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