usually on the agarose gel , the primer dimmer appers below 100 ( but its not a role )

if u make the product run for a good time u will see that the primer dimer will faint away

and the specific bands wont ,,,,

also on gel the primer dimmer band is somewhat thicker and less sharp

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What primer design software you use? I suggest using Primer3 web resource to design primers around your area of interest. It gives you only primers that have an annealing temp of 60 degree celsius, and it has been working great for me.

This is the website

RR777

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hi,to reduce primer dimer, first do a mg conc gradient , and try to reduce primer conc in total reaction volume, other wise u can also do the snap chill treatment.

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Mimmo,

I am having a similar problem with an SSR marker which has worked very well for months. I seem to be getting lots of what appear to be dimers, and the desired bands are faint or absent.

I have tried replacing with fresh primers, thinking my primers had degraded with all the freeze/thaw cycles, but am still getting the same results.

The apparent dimers lead me to believe there is a problem with the MgCl2.
A colleague told me that MgCl2 can 'go bad' on the shelf (not just in solution but in crystal form.)
Has anyone heard of this, or know how this might happen?

I got some fresh MgCl2 solution from another lab and am running the reactions now. **fingers crossed**

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[b]hello everyone can anybody tell me the exact procedure of PCR for CSF and Culture samples of TB and what is this dev primer all about ?[/b] IT'S URGENT

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make a new thread, may be then you will get some response

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From the pictures it looks like you have unspecific primers, which bind to more than one site... are you dealing with plant genomic DNA? If it's the case than maybe you want to BLAST you primers to see if they bind elsewhere in the genome. The bright bands at the bottom is almost certainly primer dimers. I would try to re-design primers for this.

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Hi I've 10 X buffer with MgCl2, the thing is I am also geting the same problem being discussed here above and i want to optimize by playing with MgCl as u described it but I can't separatly manupilate MgCl. Would it be okay if i add extra MgCl? I tried to titrate the buffer into 1.5X (that gives 2.25mM MgCl...2.25 is not commonly recommended...ya?)

c.u.

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Hi dear, wld teach me how to post picture on this site....

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Hi!
Does increasing the annealing time from 30 sec to a min or more help in product yield or it merely increases the likelihood of unspecific amplification products? Dose the formula 1min/kb of product legth for Extension time work?? Does that mean that if my desired product is 1kb then the extension time sd be 1 min?
What is the minimum amount of each primer that needs to be used!!

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