I would say its primer dimers as well.

Do you get the small band in all reactions (controls or samples)? Always with the same intensity? Is it a faint band or strong?

I know from personal experience that they can be very hard to get rid of. I would try and do a touchdown PCR and see if that would solve the problem. Also, if you have the option, try with different PCR reagents... maybe that will do it.

There are other options (google for them), like using DMSO but I'm not a big fan, it never solved the problem for me and made preparing the PCR reaction mix quite messy.

Lets us know if you got rid of the small band.

I would say its primer dimers as well.

Do you get the small band in all reactions (controls or samples)? Always with the same intensity? Is it a faint band or strong?

I know from personal experience that they can be very hard to get rid of. I would try and do a touchdown PCR and see if that would solve the problem. Also, if you have the option, try with different PCR reagents... maybe that will do it.

There are other options (google for them), like using DMSO but I'm not a big fan, it never solved the problem for me and made preparing the PCR reaction mix quite messy.

Lets us know if you got rid of the small band.

You can do one more thing ...

a negative control without a DNA to be amplified ... if you see the same band in your negative .. you can be sure that its a primer dimer or a contamination , then just exclude it ..

I agree with Ms. Kozmus. The small band you see on your gel is most likely the primer dimer. If it is indeed primer dimer, the band should be present in all reactions, including your negative control (reaction without DNA template), and the band should have equal intensity when examined under the UV. If that is the case, you should not worry about it too much.

Yes this band appear even in the negative control and in the same intensity. but the problem that i can not get the specific product. how i can solve this problem.

It's hard to get read of primer dimer which is most likely your problem. Try touchdown PCR. Sometimes it's much cheaper to order a new set of different primers instead of wasting a lot of reagents trying to fix this problem though.

Hi dear Serageldeen

You must run MgCl2 gradiant PCR and also restricted temperature gradiant PCR to find the best temperature and MgCl2 concentration with shrp bands and no small non specific bands.

Cheers

Hi Serageldeen,

I agree with the suggestion above....but if you have done this previously...do this now to see the specificatiy of your primers too........check primers on NCBI primerBLAST to see their specificity ..

bye

@Serageldeen Sultan You mean you do not get the 389bp band you were supposed to? If I were you I would first worry why I do not get the band I want rather than trying to make the primer dimers disappear. And if this is the case you need to lower the annealing temp, not increase... again, touchdown PCR should help you get a band. In the worst case scenario you do not have any template DNA as Igor Moshynskyy said. If you are sure you have template DNA (maybe try PCR with a different set of primers you know that works to confirm) and you still cant get the amplicon you want then as Mohammad Alimohammadi said, its cheaper to order a new set of primers (with different sequence, just to be clear) than to keep trying over and over and over with no success. Good luck

@Masood Sepehrimanesh With the HotStar Taq Master Mix (Qiagen) he cannot optimize Mg concentration because it is already included in the mix.

Unutilized primers in the reaction most likely to form self/hetero primer dimmers and can be extended by taq pol. Optimal primer and template concentrations may reduce primer dimers.

Dear All

I extracted the DNA from bacterial pellet and confirmed by running in gel and specto that template DNA are there as well as I tried another pair of primers but I got the same results. What are your suggestions.

You should try to first get your required product before worrying about non-specific bands. Increase MgCl2 concentration-as much as to 4.5 mM in 1X rxn and decrease the annealing temperautre-even down to 53 degrees Celsius. Also check the GC content of the region. If it is greater than 65% or so you may need a different protocol or a different enzyme.

I would suggest you to start PCR with less stringent conditions such as low annealing temperature and increased MgCl2. Check your template concentration. Even too low or too high template concentration can yield no result.

In your case, i would suggest you to do gradient pcr with annealing temperature starting from 52’C. Start with 2mM MgCl2 in the reaction.

If nothing works, reconfirm your bacterial strain.

What is Tm of region of primer that overlaps with template?? Is it having High GC content?? and how much concentraion of DNA in your 1X reaction. Try using gradient PCR and use master mix available for high GC contents

Please keep c

Tm is 64C (GC ratio 60%) for forward and 66C (GC ratio 43%)for reverse. DNA conc. is less than 1ug as kit constructions.