Hi Jui, as the band size is less (200bp), I think there can be primer non-specificity. The primers you were using might have amplified at a different region of the target. I suggest you to run an in-silico PCR and see the results. If there is any band size other than 7kb, I suggest you to optimize your reaction conditions (a very accurate annealing temperature, etc.). I hope this will help you.

Is it a bright and sharply defined amplification band or just a diffuse spot? n the latter case, these could be  the primers perhaps forming aggregates. I always run a control reaction without the enzyme. Primer aggregates are always visible in the negative reactions and usually also in the positive ones with enzyme (although here the intensity is diminished due to primer consumption).

Besides this primer band, the positive reactions sometimes show a bright amplification band with the expeected size. Other times this second band is very weak or not visible, but the mutated product can be obtained anyway after transformation.

The more intense the band is, the higher probabilities of getting colonies and mutated products. If I have problems, I electroporate with reaction products to increase transformation efficiency. If I cant get colonies or the genes are not mutated at the end, I try to change amplification conditions or redesign the primers.

Need to know: size of plasmid, annealing temp of primers, elongation time, polymerase used,  location of primer annealing versus insert location. Such a clean product suggests there may be no insert, but that depends on the above.

I havent seen the attached photo until now. These could perfectly be the primers (although its not possible to exclude a short amplification product), Do you  have any negative control reaction (w/o the enzyme or w/o the template) to be sure?

Are you doing the amplificaton of the whole plasmid for mutagenesis, is that right?

Hello, I am doing the amplification of the whole plasmid for mutagenesis. I did not run any control, would you recommend a control without template?

The plasmid is approximately 8 Kb. The primers have a Tm of 63.1 C and the annealing temp is 54.7 C. Also, I did check the primers for self dimerization via IDT,s oligoanalyzer and it was all fine. I also ran a gel for plasmid to see if it is expressed or not and that also turned to be fine. Should I increase the annealing temp ?

Peela is right to suggest in silico PCR (or simply Blast the primers against the plasmid). The primers are probably quite short so I doubt its a "primer-dimer".  I suggest that two of your controls would be to use the individual primers with all the other components. My wild guess is that you might need to redesign one of them.

Actually in this case what I would recommed is a control with template, but w/o enzyme. If your band corresponds to the primers as I think it will be present in both the positive and the negative reactions. 

Then you could transform with both the positive and the negative reactions and theoretically you should get more colonies from the positive reaction. Sometimes mutagenesis reactions do not produce visible products in an agarose gel, but are still good enough to transform bacteria and to obtain the mutated plasmids. Thats why several quickchange mutagenesis kit handbooks say- you should transform with the products even if you cannot see amplification products on a gel. Oc course, if you get visible abundant amplification products it will be easier to obtain the mutated genes at the end. So you can try to opzimize the amplification.

A control w/o template (like in any normal PCR) would also show the presence of primers bands, but its not useful as a transformation control, because the template plasmid would not be there.

Thank you all for your replies!! The primers are 33 bps long, will be visible in this gel? the ladder I used is 2 log ladder. Also, I am using phusiotaq mastermix which has taq polymerase in it already. If I have to run a control w/o enzyme then I will have to make the mastermix. I was looking for recepies for mastermix but there are varying concentration. Can you suggest me mastermix PCR reaction recepies?

If you are using an already prepared master mix, then you can only use the control w/o template. If you see the 0.2 band, its caused just by the primers. If not, you have a non-desired amplification.

for mutagenesis, use a proofreading polymerase (kapa,phusion etc.) and do the -E control, as after the PCR you will need to cleave the template by DpnI and transform. without that control, you cannot know how many resulting colonies are your backgound and can find yourself scanning WT colonies.

Interestingly, the higher your template concentration, the lower the intensity of the PCR product band. This may be indicative of non-specific primer binding or primer dimers which are producing attenuated PCR products. However, since the resultant bands are of a specific size, I would think that it is more of a technical/primer design problem. Even more so if the results are reproducible. I would suggest that you check your template sequence against your primer sequence to determine if there are any potential alternative primer binding sites, get the amplified PCR product sequenced to determine the problem or try optimising your primer annealing temperatures via a gradient PCR. Hope this helps.

If you are really expecting a 7Kb product and you see product of 0.2kb, which may be the primer dimers. Some times, you may not see the product in gel when you use Stratagene Quickchange site-directed mutagenesis kit and yet you may get positive colonies after transformation. So please clarify which kit you used for site-directed mutagenesis, how many cycles of PCR reaction you carried out etc to get relevant answer to your question.

I am using Phusion® High-Fidelity PCR Master Mix with HF Buffer and 25 PCR cycles. Tm for the primers is 63.1 C and the annealing temp is 60 C. The primers are 33 bp long.

This is most probably a primer dimer issue. It is very common when using the QuikChange primer design. I recommend redesigning your mutagenesis primers so that they will not be completely overlapping and also increase the annealing temperature up to 70 degrees, or even try the 2-step protocol for the Phusion polymerase. Do not forget to extend the time of the exension step at 72 degrees depending on the length of your plasmid (30s/kb). For the alternative primer design, please check this article (http://bmcbiotechnol.biomedcentral.com/articles/10.1186/1472-6750-8-91)

Can any of the respondents explain to me what a primer dimer "issue" is, given that the primers in this case are 33bp and the product on the gel is 200bp?

Thats why I dont use the term primer dimers, but primer aggregates to refer to them. These bands, when produced in the absence of template or of enzyme, are clearly dependent on the presence of the primers and they dissapear or diminish in positive reactions due to primer consumption, but sometimes the apparent size does not correspond to 2 primer molecules (often higher), so we cannot strictly say that they are dimers.