You indeed have an interesting question. I am surprised that the "500bp" band gave you 750bp of sequence. The only thing that I can think of is that your PCR fragment has a different conformation rather than being linear. Remember that DNA can adopt different conformations (A-DNA, B-DNA, etc.) This may happen depending on the specific sequence of your fragment. Does it have a lot of G's in a row? Or a stretch of AT's flanked by a bunch of G's and C's? Sometimes you can get your DNA to behave if you change the buffer conditions during electrophoresis. For example, if you normally run TBE gels, switch to TAE. Also, make sure that you are not partially denaturing your fragment prior to or during electrophoresis. If your sequence can partially melt, and then form hairpins, it would not run at the correct place on a gel.

However, since you sequenced the band and it has the correct sequence, you can safely ignore the position of the band in the gel.

Hi Masita,

can you give us more details?! Primer-sequences and your PCR protocol plus Master Mix protocol.

Cheers, Nadine

I am also agree with Dr. Arash.

I think, there is a possibility of non-specific amplification (primers attach to wrong place or non-specific binding and amplification). You can try, Touch-Down PCR.

When you say -

"The fragment is complete and corresponding to the sequence I want to amplify."

do you mean that you have managed to sequence 750 bases? Checked with both primers? and you are just wondering why this fragment would run alongside the 500bp mark on the ladder?

Probably you have amplified your target sequence (750 bp) with very low efficiency while most of your DNA is some random by product (500 bp). Analyze your PCR product with restriction enzymes to learn what is restriction map of product you have obtained. Certainly it is somethinh different than expected and you need to change PCR conditions and/or primers.

I am wondering whether the primers are designed on the exact species or closely related species. If it is cDNA it may be splice variant or either one of the primer should have a false priming site. Flanking regions for any gene can vary across species and that may be the reason why the product size is reduced.

Thank you all.

Sorry for not being clear, but yes, what Amanda said is correct. I managed to sequence all 750 bp from my PCR product, nothing is missing, checked with both primer, and it ran alongside 500 bp of the ladder.

I purified the 500 bp fragment before using it as the template for sequencing.

My template is a transgenic construct with primer that should amplify tail portion of the human gene promoter and head portion of mouse gene. I check with PCR in-silico, my primers don't bind anywhere else in both human and mouse.

Thanks for the suggestions. I will try checking with restriction enzyme and also touch down pcr.

You indeed have an interesting question. I am surprised that the "500bp" band gave you 750bp of sequence. The only thing that I can think of is that your PCR fragment has a different conformation rather than being linear. Remember that DNA can adopt different conformations (A-DNA, B-DNA, etc.) This may happen depending on the specific sequence of your fragment. Does it have a lot of G's in a row? Or a stretch of AT's flanked by a bunch of G's and C's? Sometimes you can get your DNA to behave if you change the buffer conditions during electrophoresis. For example, if you normally run TBE gels, switch to TAE. Also, make sure that you are not partially denaturing your fragment prior to or during electrophoresis. If your sequence can partially melt, and then form hairpins, it would not run at the correct place on a gel.

However, since you sequenced the band and it has the correct sequence, you can safely ignore the position of the band in the gel.

Just curious. What did you use to stain your gel?

some stains do affect DNA mobility when added to the agarose preparation.

Instead, try running your gel without any stain (either in the agarose or the running buffer) and post-stain your gel after running it. This may be the reason for the "apparent" shorter product length.

@David Yes. Many Gs and Cs in a row. I run my gel using 1x TAE buffer. Is it going to help if I increase the denaturing temperature during PCR cycle?

@Eric I use EtBr mixed in the gel.

Again, thank you very much for all of your valuable insight. I will recheck my sequences again to be sure and do additional experiment to confirm. It's very confusing for me but I gained another experience.

Sorry, I was wrong when I said I have all 750 bp of my target sequence. I didn't thoroughly analyze it.

I have discussed this matter with my PI and he pointed out the problem. It turned out there is another similar sequence with my reverse primer so the primer annealed to the unexpected site resulting in shortened PCR product. Fortunately, I still can use this primer set since the shortened product still have my main target.

This is a very valuable lesson for me. I need to be more careful in my analysis.

Probably the PCR primers is amplifying a smaller segment spanning your target region in the gene. As you mentioned if you extract and sequence the PCR band of 500 bp you will know that it is a smaller product which could have included your target region. However this 500 bp product is the predominant one and not the expected 750 bp. Try a touch down PCR as suggested to get an increased yield of 750 bp product. Else design new sets of primers spanning your target region. Make sure the product is smaller in size that 750 bp.