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Possible primer dimer problem..Need solution!!!


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#1 astroboy89

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Posted 15 September 2014 - 11:33 AM

I have designed primers to isolate a promoter from rice.I used the Life Technologies OligoPerfect™ Designer. 

 

I obtained the following primers:

 

1) Forward Primer : 5'-T T T T G A A T G C T G G A A T G A T A A G C-3'

2) Reverse Primer :  5'-C A C T G A A G A T A T A A T C T A A C T A G C T A G C T A -3'

 

On analyzing the reverse primer in IDT oligoanalyzer, I found this in self dimer analysis

 image.jpg

 

 

On using 5' overhangs containing RE sites for cloning , in the reverse primer the IDT Oligoanalyzer showed this during self dimer analysis:

image.jpg

 

 

Now what should I do if I decrease the length of the reverse primer, the Tm difference between the primer pairs increase more than 5 C.What should I do?

I know the formation of primer dimers is inevitable.

 

Is it possible that I first preheat the primer and water at 95 C for 5-7 mins and then add the rest from the master mix.Will it affect the Taq polymerase?

 

I cant afford to buy Hot Start Taq polymerase now.Can I do the same thing with regular Taq?

 

What should I do? Please help.

 

I havent ordered the primers yet..



#2 bob1

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Posted 15 September 2014 - 01:16 PM

If you need these primers to start exactly where you have them now, then you don't have much option as to the primers other than to extend/reduce them a base or two at either end. If you can shift them, then you have some choice.

 

Don't worry too much about self-complementarity, you should be more worried about inter-primer complementarity.  You can reduce secondary structure in your PCR by the addition of Betaine or DMSO, which should eliminate any problems you are having.  5 degrees is not a huge difference in annealing temperature, I have used over 15 degrees difference successfully.  Note also that any additions to the primers in terms of restriction sites etc, don't play any role in the annealing temperature calculations!

 

However the best thing you can do is order the primers and try them in a conventional PCR.  Play with the annealing temps a bit to see if they work. If they don't work at all, then you may need to re-design.



#3 phage434

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Posted 15 September 2014 - 01:39 PM

I disagree with Bob1. I think these primers are hopeless in their current form. Why don't you extend them a few additional bases. The fact that they bind tightly is not really the problem. The problem is that they bind tightly at the 3' end. Even two bases which are different at the 3' end will go a long way to solving your problems. It would be ideal if they were both purines (G or A) which will mismatch more forcefully than pyrimidines.

 

Exact or near exact matches at the 3' end make it possible to extend the end. Once this happens, the primer no longer matches the target, but matches the dimer better. Amplified problem.






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