Hi, your situation sounds very interesting because usually we would verify the PCR products by conventional gel electrophoresis before we do sanger sequencing.

Perhaps you have got some PCR products on the gel but the band was too faint that it couldn't be noticed by naked eye?

Zx Chong My first thought was that the concentration was indeed too low to be seen on gel. But it's kinda problematic since we can't afford to do sanger sequencing on all samples that seems to be negative on gel electrophoresis. it will take too much work.

I'm currently searching how to know the minimum concentration of amplification product to perform sanger sequencing on. My guess is that the ability of the system to obtain a sequence (signal) is greater that the ability to obtain a fluorescent signal on gel electrophoresis. I don't know if I'm clear enought.

Anyway, thank you very much for taking interest in my question.

did your ladder look good on the gel and was the pcr product likely to stay on the gel according to the ladder bands? clearly I am asking did the band run off the gel and was there enough ETBR to see the band. The sequencing reaction is an amplification step so if you run more cycles than the kit suggests eg 35 cycles then it will amplify a good signal on very little template and the sequence signal can be measured over about 2 orders of magnitude so again,with a low background very small signal strengths can give a good sequence. With this type of question I always phoned the tech support people at Applied Biosystems who were always very helpful ( presumably now Thermo as ABI were taken over some years ago

Paul Rutland The ladder looked good, the band did not run off the gel, and there was enough ETBR to see the band, cause i had another sample positive on the gel and also my positive control was perfectly visible. I think your second point is valid, probably my signal was too low to be seen on gel, but after the sequencing reaction it got amplified and we were able to get a decent signal on sequence reading.

Thanks for your help

in that case as a start to your calculations approximately you can see 10ng dna in ETBR on an agarose gel with small wells and not run too far. You sequence 25ng X ( dna length in Kb) so if your amplimer is 200 bases then template is 25x 0.2 = 5ng So I suggest you may have 2ng dna on your gel ( not visible) so if you can increase the sequencing signal by a factor of 2.5 by running more sequencing cycles you should get a similar signal to sequencing 5ng. This means only 2-3 extra cycles of sequencing to get a similar signal intensity. You should do this by decreasing the sequencing volume to keep the reagent concentrations high so the reaction kinetics remain similar and running more than the 25 cycles recommended by ABI in their kits

Dear Marc-Antoine,

Yes, sometimes it is possible to get good sequencing results, without a visible signal on electrophoresis gels.

In the past, I saw similar relationships with commercial HLA typing and sequencing kits.

- Perhaps you could use acrylamide gels together with silver staining, to make your PCR products visible?

Be happy! :-)

You found the expected sequences!

Best regards,

Thomas