PCR band slightly BELOW expected length ?! - (Jan/14/2013 )

Hi people,

I did a gradient PCR with several annealing temperatures and samples. The expected product length would be 200 bp. In all of my samples, regardless of temperature, I got the same single band (no smear etc.). However, this band is slightly below 200 bp, definitely !

Do you think this is the real product (but why would it be below the expected length then ?) Or how should I interpret this ?

Thanks for your help ! This issue is very important to me.....

Did you BLAST your primers before you sent them off to be made? If not - there may be another binding site in the RNA or the genome somewhere that could cause this.

and how did you check it? On a standard agarose gel? and how much is slightly? A size determination on such an agarose gel is not very exact (especially with smaller bad sizes) and also other factors can influence the results (e.g. DNA amount, gel matrix heterogeneity, electric field differences)...
Perhaps you should check it twice if the same difference occurs again.

@bob1: PrimerBLAST only gave the correct target as a result for this primer pair; besides, I took them off a publication where they have been used successfully.

@hobglobin: 2 % agarose gel. As for how much "slightly" is, I find it difficult to describe in words, I'll try to post a photo here.

Here is the gel photo. Sorry for the bad quality, I think the bands are visible though. So the marker right "above" the PCR prduct band is 200 bp, underneath its 75 bp.



What do you think about it ?

and which one is the slightly smaller one? I could not really identify it...perhaps the far right one?

@Tabaluga: Did you do a negative control? Does it have the same band? (in that case - dimers)

@Trof: I did a H2O control, nothing to see there.

@hobglobin: not sure if I understood your question correctly, but actually all of the samples have the same band, which is slightly smaller than the 200 bp marker.

Other bands of different PCRs are having the correct length? There is still possibility to sequence the product, to be sure what it is.

and I got it somehow wrong (thought only one of several is below 200 bp)...anyway did you ever had this then with other samples? Or is it from sequence data just expected or calculated (only)?