How to get rid of PCR primer dimer - (Mar/17/2003 )
What mostly affects PCR specificity is :
1) annealing temp ( the higher gives the most specific amplification)
2) the MgCl2 conc. is the most important above all : the higher it is the less specific will be the amplification
3) the annealing time : the longer it is, the most specific will be your amplification, because false pairing will tend to be disrupted with time.
And if increase of Tm, decrease of salt, extension of annealing time don't work, then try the touch down PCR:
start your PCR cycles with high TM and go down incrementally at each cycle ( decrease TM of 2 to 4°C every 2 to 3 cycles)
So that U only do specifc priming. It may be the best in your case. Start at above your normal Tm. like 60 or 65°C and finish down to it like 50 or 45°C.
Thanks for the suggestion. Will try a touch down pcr. I have 100pmol/microlitre primer conc. how much of it sd be just enough so that i dont get a primer dimer?
1) annealing temp ( the higher gives the most specific amplification)
2) the MgCl2 conc. is the most important above all : the higher it is the less specific will be the amplification
3) the annealing time : the longer it is, the most specific will be your amplification, because false pairing will tend to be disrupted with time.
And if increase of Tm, decrease of salt, extension of annealing time don't work, then try the touch down PCR:
start your PCR cycles with high TM and go down incrementally at each cycle ( decrease TM of 2 to 4°C every 2 to 3 cycles)
So that U only do specifc priming. It may be the best in your case. Start at above your normal Tm. like 60 or 65°C and finish down to it like 50 or 45°C.
the appearance of primer dimer is mainly due to the overhigh primer concentration or content .so the best wey to avoid it is to reduce the primer concentration and content
i used PCR additives consist of 10% glycerol, 10% DMSO and 10% Formamide. Add to final concentration 1% for GC rich PCR.
Hi guys
I am also having the same problem! I am trying to amplify exon 4 of the caprine kappa-casein gene with primers I got from another article. 2 months ago I tested the PCR reaction with the temperatures they gave (Ta = 63). It worked ok. A colleague suggested that it would work better at Ta=67. I tried that - it worked beautifully!!!
Now, I want to integrate the PCr step into the rest of the steps and suddenly at Ta=67 there is nothing but a few weak bands and hectically bright primer-dimers! I know what you're thinking: old ingredients, or it must be some mistake. Not so. I tested it again carefully at 67 - no luck. Tested it with new primers and new dNTPs and new water - no luck. Decided to play around with the temperature - tried 65 (so-so), 68 (nothing), 62 (so-so, less specific bands and still have primer-dimer). Now I dont know what to do. I dont know if I should start messing around with MgCl2 concentration because it DID work. Someone mentioned that my DNA may have degraded from being stored in the fridge? Any help will be greatly appreciated as I dont have any more hair left to pull out!!
Robyn
Hi,
I have one problem of cloning primer. I designed the primer for 1200 bp coding region but i couldn't get my required size when i performed PCR . I changed all the conditions and increased annealing temperature and touch down PCR . Even i sent the sequencing for the another clear band even though that was not my target.
Please give me some suggestion.
regards,
learned a lot !
for me, reducing the primer concentration is the best way to reduce the primer dimer on the gel. after a lot of trial, I found 10 pmol of primer concentration give me no primer dimer in all my PCR reaction on the gel.
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**
Hi there,
I have started to get a bit fed up after trying to get an unspecific product for a primer set without luck. I tried optimization by trying gradient PCR from 48 to 58 ° C but all it showed was smeared band, which seemed to be because of unspecific binding. I though i might repeat with the same. I did so and still the same result. I am thinking, it might also be primer dimers. I am now running a re(-re) optimization in a range of 55 - 65 °C. The Tm of both the primers is 57.3°C.
My question is - how can i try changing MgCl2++ conc. if i m not using it separately in the master mix. MgCl2++ is contained in the 10x buffer so can i add some MgCl2 in it or what?
Finally, i tried
The MOST important thing to try with primer-dimers is to redesign the primer set. I can't imagine spending a month optimizing barely working primers. I strongly suggest that this should be your 2-day frustration action. I'd also recommend PCR master mixes -- they avoid the temptation and inconsistency of fooling around with a half-dozen components and being unable to reproduce working behavior. Try the Finnzymes (NEB) Phusion master mixes and have stuff just work (or not, but at least with little ambiguity).