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?
doesnt the annealing temp is supposed to be at least 2 degrees lower than the Tm??
try to use 4% agarose gel with TBE buffer to analyze your PCR product! if PCR is successful, you might separated your PCR product from primer dimer!
thanks far all your advices I am used to check everyday.
I am working in marker assisted selection in vegetables....but let's briefly try to switch to the Big Problem....
I set up a CAPS marker for fruit shape, it worked nicely for months....than the first problems started to get me insane...
this is how the gel looked months ago (it's after digestion, but anyway is clearly visible a smooth aspecific below the main band, the 900 bp one, undigested, but was OK)
this is what happens now (only PCR, no digestion):
shitty bands, tha main one looks as a double one, aspecific at the bottom (or primer dimers? it seems to me too long for primers)
That is what happened this morning:
it's a test for primers titration and mg titration, three dna samples repeated three times; let's look at the one on the right side (the highest Mg conc)
the band below the main one became more faint, but it's still on....the 100 bp band is extremely strong....
How does this story sounds to you?
from the first period, in which everything was working, we changed everything, the reagents works in pcr of other fragments, as well as the template dna...the primers have been ordered new two times......
thanks for your help......
PS: I apologize if I made some mistakes in posting images...
In our experience, you'll always get these unspecific bands in the apoE genotyping unless you do a nested PCR. IE you do like 12-15 cycles of PCR including 5%DMSO from the genomic DNA with one set of primers, then take 1ul of the product, dilute it 1:20 and use 2ul of that as template in another PCR reaction with a set of primers that is inside the first set.
Hope that helps
Improved method for genotyping apolipoprotein E polymorphisms by a PCR-based assay simultaniously utilizing two distinct restriction sites (Clinical Chemistry).
I use formamide with DNA sequencing for denaturing the DNA, but if you use it with PCR, is it still posible to let the primers anneal?
What is the reasen to use DMSO in the PCR reaction?
u can try touch down PCR. Problem with primer dimer can be overcome optimising lower concentration of magnesium chloride, using higer concen of templates and DMSO 2 to not more than10%. for higher GC content, u can add glycerol.
I definitely agree with subarna. Touchdown solved all of my problems, either with or without DMSO or betaine. The presence of the 100 bp product is simply due to mispriming close to one of the ends, which is more efficient than full-length product (compare 100bp with 1500 bp...)
Start at least 5 C above theoretical Tm. That way, you'll only get extension from 100% identical sequences. If you still get the 100 bp product, then you clearly have a repeatingsequence, and you'll need to redesign the primers.
i am new to this site , and have found it useful for my work. i am facing a problem regarding PCR amplification. i am trying to amplify Prot A gene from pRIT2T vector . when checked in the gel , the pcr product getting only primer dimer band, there is no expected band seen. When i do the same reaction mixtures used in other pcr machine getting expected result. why i am not getting with my pcr. can you please help me in this regard??
It looks as though there is a problem with the first machine. When was it last serviced? Have others had similar problems?
hi some one
please tell me exactly how to distinguish a primer dimer in a PCR gel. does all the bands and smears below 100 bp be considered as primer dimer