How to get rid of PCR primer dimer - (Mar/17/2003 )
I also had less than 50 bp band and I thought that it could be primer dimer, although I didn't see such sharp bands before 2 weeks while using the same primers. Tm of my primers are 50.5 and 54.2... annealing temp is 60 °C. so should I try lowering the annealing temp and increasing the template amount and changing my primer aliqout with new one?
I was having the same problem with primer dimers, and I followed ocean recomendation in using DMSO, and it really worked!! For me it worked the best at 5%DMSO.
One thing to also try is designing your primers with AA on the 3' end.
Regarding Mg2+…too few ions will cause low product yield while too many will result in non-specific products and promote misincorporation.
Lower Mg2+ concentrations are important if you are worried about the fidelity of your DNA synthesis. The recommended range of MgCl2 concentration is 1-4mM. You’ll need to raise the Mg2+ concentration if your samples contain EDTA or other chelators. Good luck!
Hi, I was also having some trouble with primer dimer just recently trying to amplify genomic DNA. I tried a few things:
1) reduced primer concentration from 0.5uM to 0.25uM
2) reduced amount of initial template used in PCR (from 50ng to 25ng)
3) added 2% DMSO to the mastermix
...and voila....primer dimer disappeared. Thanks everyone for all the different suggestions.
Number 1 peice of advice is to use a program to design primers!
I can not stress this enough. My favorite is the free online primer design program, primer3:
For Primer Dimer proplem the best is the HOT START technique (Don't add taq before tubes reach 95 temp)
OK I am having very very strange dimer problems.
Using the BioRad iQ Sybr mix I get no primer dimers (Ta=60C). However apon switching to immolase I am getting significant primer dimer, which increases with increasing Ta (62C and 64C). The dimers are also more of a problem with lowering amounts of target DNA. Increasing [Mg++] lowers primer dimer!
With immolase I use 2 uM primers but with the BioRad mix I use 5 uM. Also the Sybr we use is diluted in DMSO so the final concentration of DMSO in our reactions is 5%.
I would prefer to use immolase as I am doing comparative quantitation and it gives more consistant amplification.
Usually fragments as big as 1.5kb are little bit harder to get working in PCR. If you are using chromosolmal DNA, try taking a plasmid DNA if you have one created already with your fragment in, usually works much better than from chromosomal DNA. You can also try diluting 1:10 your O/N culture, heating at 95 celsius for 5 min and then using 5microL for the PCR. Good luck!
i was encountering wid same primer dimer problem and in shortly i am going to design SSR primers using Primer 3 output software. please can anyone suggest me the conditions or the necessary parameters required to avoid primer dimers and to get a ideal PCR product. please reply soon.