1. David Oppenheimer added an answer June 23, 2014

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.

2. Chandrasekhar Natarajan added an answer June 23, 2014

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.

3. Debanjan Bhattacharya added an answer September 17, 2019

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.

https://www.researchgate.net/post/Why_is_my_PCR_band_size_less_than_expected

Thanks Kannan

Hi Beatrice,

In addition to @Mani Kannan's answer, I would suggest checking the integrity of the target primers for contamination (hence amplification in the NTC), and also to see if they are perhaps breaking down thus lowering the actual primer concentration (hence faint bands).

Also check for contamination on the other common reagents.

Best,

Fiona

Hi @Fiona Nelima Mumoki Ok I will do so.

Thanks

Thanks you all for your kind responses. I found out from the manufacturer that the problem may be due to damage or malfunctioning of the column type W tubes I received from another lab. So, I tried with the column type W tubes from a new kit and it worked.

Cheers,

Beatrice