Hi everyone,
Can anyone give me advice about how to improve my qPCR primer efficiency: I design primers using primer3 trying to get GC content around 55%. When I perform primer test using standard curve I see for some of them they are not working efficiently at 60 degrees. I am trying to play with the temperature - if efficiency is low, I decrease normally T and if high - rise but for sure it would be much better to have primer database that all work at 60 degrees. Any tips, please
Dear Elena,
The best option is to redesign your qPCR primers using this time the fellowing database: http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHome. Pay attention to the temperature which should be close to 60C, amplified fragment length between 100 and 150bp. Also double check if your primers are specific or not, for that you can blast them in many databases like NCBI or phytosome.net.
Good luck,
Dr. NL
Hi Elena,
Dr Lakhssassi is correct, NCBI Primer blast is a easier option. Select all the parameters that you want like annealing temp, size of the product, exon or intron spaning etc. Primer blast gives you several results choose the primer pair with fits the general criteria for good primers. You may want to make atleast 2-3 primers for a gene, as not all primer turn out to be efficient. Since you are changing the annealing temp post your results, it would be better if you cloud perform a temp gradient for doing the standard curve. All the best.
Regards
Prasad.
I agree that a re-design of the primers is in order -- especially if you get low efficiency and a bad melt curve. In addition to the previously mentioned resources, IDT has a very user-friendly primer design tool for qPCR. It can select primer pairs that flank an intron in order to improve cDNA vs gDNA specificity. It's optimized for TaqMan-style qPCR, but the primers work well for SYBR-green based qPCR as well.
https://www.idtdna.com/scitools/Applications/RealTimePCR/
Hi guys,
Thank you for suggestions. My primers are okay in terms of specificity, GC content and product size, I check the melting curve by in silico PCR using Umelt software (very nice one if you haven't used it), but unfortunately when you perform primer test they don't have 90-110% efficiency. Probably I should just take it philosophically and test other pairs of primers. I was just wondering if there are some tricks to get nice primers at the first strike.
Have you a lot and have a nice day!
Dear Elena,
before changing the annealing temp and design new primer, I would suggest to optimize the primer concentration of your PCR. On basis of my experience an increased primer conc. (sometimes also decreased) has a big effect on reaction efficiency and formation of additional products.
If you want to create new primer pairs, you should go on using Primer3 in my opinion. NCBI primer blast is easy to use, but with Primer3 you have much more possibilities to get different primer pairs and define the primer position in detail.
You could try to design primer pairs for different parts of your gene of interest. Especially in bigger genes, different parts may be differentially represented in your cDNA, which alters signal intensity and reaction efficiency.
Did you solve the problem??
was it a question of testing different primer concentration combinations?
Primer3plus looks a very convenient tool:
http://primer3plus.com/cgi-bin/dev/primer3plus.cgi
I did a qPCR. One of my primer pair had efficiency 105% . But I am observing that the Housekeeping gene I used (ldh) and my gene of interest are expressing late(after 28 cycles). Other Housekeeping gene (imp2) expressed within 25 cycles. What can be the reason?
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