during PCR for gradient I used 55C to 68C annealing temperature. Tm of forward and reverse primers was 65.3C and 59.4C respectively. suggest me what can I do? specific product is ~436bp and non specific product is ~530-550bp.
Hi Madeeha, this may occur when the annealing temperature is too low, or when the primers concentration is too high.
To optimize the PCR annealing temperature you should have also that of the product and use this formula:
Ta Opt = 0.3 x (Tm of primer) + 0.7 x (Tm of product) – 14.9
PCR optimization reference paper attached below.
Hello, Madeeha Tariq
You can reduce/overcome the non-specific band by optimizing the annealing temperature even lower than you have used in earlier studies. It may be started from 48 C to 55 C.
Secondly, you can use DMSO as an enhancer to increase the specificity of your primer to the target DNA site.
other Suggestion,
Sometimes excessive extension time can allow nonspecific amplification. Generally, use an extension time of 1 min/kb or an annealing time of 30 sec.
May this will help you
You must check the extracted DNA (must be not contaminated) and it's purity, PCR conditions must be optimize, you must use specific primers, volumes and concentrations in PCR reaction mixture must be also checked.
It would be useful to see the picture of the gradient pcr experimentm To minimise non specific bands you could try a gradient of DMSO concentrations 1% to 10% dmso and see if the band vanishes. Also check the primer pair using primer blast as they may simply be properly amplifying another sequence in which case primer redesign may be needed. Is the thick band diffuse and smaller than the proper band in which case it may be primer dimer and the answer may be to use less primer to clean up the amplification. A picture would help in this situation
Mostly this occurs due to annealing temperature! Try adjusting the temp, likewise make sure you are dealing with purified products,as purity is of essence in movement of products across the gel... hope you find this helpful.
Confirm first whether the thick band appeared due to contamination by running negative control.
If no contamination occurs, try running gradient pcr starting the annealing temperature 3°C below the low Tm of primer. In your case, low Tm is your reverse primer 59.4°C so start gradient Ta from 56.4°C to 54.4°C (-3°C to -5°C below Tm). Usually i start 3°C below and it worked for me most of the time. Ta you used is very high which more than 59.4°C that exceeds the Tm. Ta should always be lower than Tm.
Thank you for the images Madeeha Tariq . Clearly this is not a primer dimer problem. You can try a dmso gradient at 57c annealing temperature but the larger band is very strong and it is probable that your primers are annealing better to another sequence. Possibly a pseudogene or other member of a gene family. If this is the case then redesigning the primers and checking the specificity with Primer Blast. Also check again that your primers should amplify the smaller product by blasting the primers because the larger band is a much stronger amplification
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