You will need to run your PCR on a gel for long enough to separate your specific product from the primer band. Excise the band and purify your PCR product from the gel. You can then continue with your cloning without worrying about primers.

From my experience, to avoid primer dimer to should:

1-try to design both primers without complementary regions

2- not put both primers together at the same time, meaning that you add one primer at the time when making the master mix

I hope that could help you

Hey Suraj,

Please give us the main parameters of your PCR/clean up procedure. Length of amplicon, number of cycles, primer length, amount of template, GC content of primers and amplicon.

Also please post a picture of your gels a) prior to clean up ("raw PCR amplicon"), and b) after clean up.

Stefanie

If u see primer dimer on ur gel and its bond is thick soyou should chek ur primer disigning, but if its after clean up its not important

Hi Suraj!, one alternative is to start all over designing the primers again. One very good tool to evaluate if your primers will form primer dimers is autodimer (http://yellow.nist.gov:8444/dnaAnalysis/primerToolsPage.do). You can check if your actual primers are complementary.

The second option is to check the clean up protocol. If you are using column kits you should check the amplicon size that the column can catch. Some of them suggest use isopropanol to catch small amplicons like fermentas genejet purification kit. So if you avoid to use isopropanol in the binding step you should get only bigger amplicons. How big is your amplicon?

The third option is, maybe you are not getting primers but unspecific amplicons instead. How big is your primer dimer band? you should check that before to proceed.

Regards and good luck! :D

Hello suraj,

To avoid primer dimer formation, the above suggestions can be considered, and in addition i put few words here:

1. Decrease Primer concentration during PCR , you can play with different concentrations and see the difference.

2. Vary MgCl concentration

3. Increase annealing temperature and for longer time also

4. Use DMSO may be upto 5%.

5. Increase DNA template amount

Otherwise you have to go back to primer designing (primers with less complementary bases).

Good luck.

thank you all for your responses.

Here are the details of my PCR:

Amplicon length: 465 bp.

Forward Primer length: 46 (with histag), reverse primer length: 30

No. of PCR cycles: 30, Amt of template used: 2.5ng/ml (as with 2 ng/ml, amplification was not coming previously).

I have tried different conc of primers (0.5-1 µg/ml).

the dimers are present before PCR clean up and after clean up also.

Thanks. How do you clean up your PCR amplicon?

Stefanie

You will need to run your PCR on a gel for long enough to separate your specific product from the primer band. Excise the band and purify your PCR product from the gel. You can then continue with your cloning without worrying about primers.

Use ampureXP beads if your primer dimer is around 50-60 bp. The size difference is sufficiently large.

@stefanie, I am using quiagen PCR purification kit for the clean up

@Amanda and @Suraj, I think you stated the all the steps for isolating a DNA fragments but I just wanted to add that he have to clone and sequence his fragments before proceeding to be sure that he have what he looking for.

Good luck for you

Hi Suraj

In order to reduce the amount of primer dimers co-purified reduce the amount of binding buffer added to you PCR mixture. I cannot say for certain how much you have to dilute the binding buffer (varies between kits) but check out NucleoSpins protocol for suggestions.

http://www.mn-net.com/Portals/8/attachments/Redakteure_Bio/Protocols/DNA%20clean-up/UM_PCRcleanup_Gelex_NSGelPCR.pdf

I trust that you have remembered to increase the annealing temperature of your PCR when using Phusion Polymerase

Good luck

Jesper

The best, as Amanda stated is to do the PCR, run the gel, cut exactly the right band and perform purification using a commercial kit designed to purify gel-band. Vivantis Technologies (http://www.vivantechnologies.com) has a kit that is designed for both PCR product and gel-band

@amanda and @abdemalik: Thanks for your responses. I have done that too. but after gel extraction, the concentration of my insert get decreased so that i can't even set the digestion rkn with that. may be I need to set the PCR for a large quantity of reaction mixture.

You could try using a hot start PCR which should decrease PD formation, other suggestions include: increasing the annealing temperature, decreasing primer concentration or increasing template concentration (which i think have been mentioned before).

If none of these work, check your primer sequences to make sure there are few complimentary bases between them/ within them to avoid PD and secondary structure (ie stem loop) formation respectively.

In saying that i agree with Amanda, cut out the exact band you need, purify the DNA then proceed with further applications.

I hope this helps.

hey there,

What you can also try is to make you PCR using tow annealing temperatures:

start with the lower one (the one that you've been using until now)- make 5 cycles like this, then, in the remain reaction (let's say 30-35 cycles) higher the temperature to the max (70-71C), in this case, the PCR will be more specific to your template and you will not loose the amplification because you start with the temp that was suitable and then when the primers made some progression the "new" template will contain the tail of you primers and you can easily increase the temperature..

Good Luck !

a hot-inactivation enzyme will help reduce the primer dimer before PCR.