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PCR Primer Dimers Troubleshooting

PCR Primer Dimers Troubleshooting - PCR - Polymerase Chain Reaction Forum

PCR Primer Dimers Troubleshooting - PCR - Polymerase Chain Reaction Forum. Discuss and ask questions about PCR troubleshooting, PCR protocols and methods, PCR products, and PCR theory.


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  #1  
Old 08-29-2008, 12:37 AM
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Lightbulb PCR Primer Dimers Troubleshooting



Please i need the help in my problem:
i design primer but when i made PCR, i found may be formation to
primer dimer under the louding dye but the band of primer dimer under
the negative control ( without RNA ) was small in size than the band
under the sample ( more thick and may be 2 band more closed ). i
belived fond 2 band in sample because the negative control and sample
contain same band in the below but found band upper it in the sample
( may be more thickness )at the same conditions. i tried at 47, 52,
55oC annealing. all tempreature give same result with diffrence in 47
the band under negative control more faint but at 55 the band under
sample more thick and light.
please, i need to know this difference meanse any thing or the
difference in size between the band under control and sample don't
means any thing and the 2 bands are primer dimer only.
Thanks for you
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  #2  
Old 09-04-2008, 09:59 PM
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Default Re: PCR Primer Dimers Troubleshooting

Hello primer dimers could be several problems:

1) too much primer added. How much primer did you add? Concentration of primers?

2) primer design issues - there may be self-annealing of the primers to themselves. You can check the two sequences if they self-anneal here:
http://www.idtdna.com/analyzer/Appli...OligoAnalyzer/

Or they can anneal to each other. This can be checked with primer complementarity with DNAMAN:
http://www.lynnon.com/

Please someone let me know if there is a free software version to check this


These are usually the 2 main ones
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Old 09-06-2008, 02:33 PM
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Default Re: PCR Primer Dimers Troubleshooting

add 4 microliter primer (10 microliter )
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Old 09-09-2008, 11:11 PM
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Default Re: PCR Primer Dimers Troubleshooting

YTNTgggCNCYNACNgCN F- primer
ggNCTRATRCCNACCTTRATR R- primer
my conc. of primer 10 micro mol and add 4 micro liter for reaction (4 F & 4 R)
please help me this is primer dimer or not
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Old 01-07-2012, 05:35 AM
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Default Re: PCR Primer Dimers Troubleshooting

I am also doing real-time PCR and I use STRATAGENE PCR. I do not see the primer-dimer formation, however, I think there is still primer dimer formation in my reaction. Because, when I am constructing standard curve I find that when I remove the lower dilution, the efficiency of my PCR shoots to 110% which is most desirable result and most accetable, while if I include that last dilution as well, the PCR efficiency shoots to 165% which I think is because of over estimation of amplification data which is because of primer-dimer in the lowest concentration. I believe that in lowest concentration, my primer doesn't find much template to bind, so there is higher chance that they will anneal with each other and second thing is that I am using primer for telomere which is repetative sequence of TTAGGG and nothing else. This sequence is all that is repeated and it is completely different than other genes. So, I think in this kind of sequence it is more favorable chance of formation of primer-dimer. What do you think?

I also see amplification in my NTC. I made fresh dilution of everything after autoclaving, but still there is amplification in NTC, which I think its because of primer-dimer and nothing else. I am using primer of concentration 2 micro molar in final single reaction volume of 20 micro liter. Do you think its primer-dimer and if I will reduce the concentration of primer, it will make reaction better?


Please kind send in me your suggestion. Waiting for your suggestion.

Yours sincerely,
Rajesh Chaudhary,
Thailand.
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