@Hanan mohammed kamal Abd el fatah Osman

Template DNA and PCr product. Both when use PCR template for run PCR(and primer binds to it), and DNA maded during PCR.

Just guessing but may it be possible that your tenplate DNA would be contaminated with a larger DNA?

You could find it out if you loaded your template DNA into another well.

Besides, a non-template control is always advisable in your gel. With that you could know if primer multimers or reagent contamination are occuring which may also explain your puzzling result.

Please let us know if you solve this issue!

Best,

D

I think, this might be contamination, DNA marker/ ladder is not clear even

I think there is an issue with your PCR(assuming the third lane with high molecular weight signal close to the well is your test sample), seems you loaded too much template that inhibited the reaction. You might need to quantify your template DNA and then dilute it appropriately.

I suggest you to load less of amount of sample and check for any possibilities of contamination. Please do mention the concentration of gel you are using while you are listing your queries.

Best,

KG

@Kaustuv Ghosh Agarose gel , 2% used.

Is solubilization of the genomic DNA guaranteed?

The Bright band is just below the well and seems to be genomic DNA remained un amplified during PCR. It could be due to very high concentration og template DNA in PCR. You can run your template DNA before amplification in agarose gel to see quality of template. Also go for quantification of DNA and its purity by UV or nano drop.

I think probably you had insert too much of your template during PCR, what is the concentration of template that you are using?

Also your ladder is not clear.

Hi

Reduce Agarose gel

I see several problems beside the gel appearance.

You say that PCR template (is that the input DNA what you mean?) is 99 bp. Why then you used such a PCR marker for it? better use a clear 100bp fragment indicator one, such as Sigma D7808 (just an example), of course dependding on your gel concentration.

What relevance is that you digested both "template " and PCR product (it is called amplicon) with Exonuclease III?

Even the first lane shows incomplete digestion (faint bands still visible).

Third lane is indeed large genomic DNA which is much up than the marker's 750 bp band. You have DNA remaining in the well, even. Which shows that your amplification was unsuccessful for the 99bp target, and you've started from whole DNA.

I think the whole experiment is unclear to you in the first place. Make it clear and then come back for some help, before doing this and that, and confusing things more.

Looks like you have several issues (your ladder is too faint and smeared to be useful), I don't think you ran the gel for long enough to get good separations, etc.

But, my main concern is that I have no idea what question you are trying to ask with this experiment. An exonuclease will completely digest and get rid of your DNA/PCR/whatever your original template is.