I have designed primers for pcr of blood dna, but the problem is the primers are showing multiple bands after doing pcr. These are self designed primers. Why is this problem occurring?
This problem can be caused by many factors.
To what gene/region are your primers designed for? Is this gene/region repeated throughout the genome? Did you run a blast search/primer-blast for these primers?
Could you provide the photo you took of the electrophoresis gel? How many bands per lane?
Make sure primers are specific to that region. Perhaps this could be due to the same primer annealing in different regions and producing amplicons of different sizes.
It is also good practice to have a known positive control - a known sequence that contains the region of interest, the annealing position for the primer, and forms bands with which you can compare.
I'll be waiting for updates. Good luck.
While you look at primer3 and primer blast software which help to make good primers you could try making the primers that you do have work better by increasing the annealing temperature and running a gradient of dmso from 0 to 8% final concentration which can sometimes cause a slightly mismatched primer pair to fail while allowing perfect homology primers to work
Primer 3 software used in designing this gene, the name of the gene is PPRG gene, the region is Exon region and I have the picture of the band, Can I send it to you in your inbox?
yes and please include the amount of dna that you use in the pcr reaction
40ng is a good amount but using too much dna template leads to more non specific bands in a pcr. If your lab has a gradient pcr machine then run a gradient of about Tm minus 5c to Tm plus5C to help clean up the reaction
If your gradient went so high that the pcr stopped working then choose an annealing temp that works +/- 2c and run a dmso ( any grade of dmso will do) with a dna that works have tubes with 1%, 2%, 3% up to 8%dmso and see if that cleans up the pcr enough to use the results. It may be that you chose one gene of a similar sequence gene family in which case primer design may have to change
It Is probably a bit late but can you post a picture of the pcr gel please Adnan Sayeed ?
take a look here, it may help:
https://www.bio-rad.com/pt-br/applications-technologies/pcr-troubleshooting?ID=LUSO3HC4S#gel2
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