I performed a PCR with a gradient of temperatures and ran the gel electrophoresis.
There were bands present at each of the temperatures (lets say 59 and 55 degrees)
I received the sangar sequencing chromatograms and there are almost no peaks, the sequencing appears to have failed for the 59 degree PCR. However the 55 degree PCR has clear peaks.
Other than something going wrong at the seqencing centre, what are other possible reasons you could think of for there to be no peaks despite having gel bands corresponding to both temperatures?
Hi,
First, why you try in two different temperature? Annealing temperature is depend on the primer(s) that you designed, it not necessary to get two bands for two different temperatures. Normally we do the gradient PCR for PCR optimization. If you got band at 55, then proceed with this temperature. You can see the different when your run the electrophoresis, one is brighter and another is less brighter from PCR products from two different temperature.
Second, PCR purification, when your clean up your PCR product, make sure the concentration of PCR products are sufficient for sequencing, some times the PCR product purification can lost some or all your DNA. My suggestion is run many tubes, then elute into single tube (same temperature into same tube), so you can increase the concentration of your DNA. At the same time, how you purified the DNA, so far when I use kit no problem for PCR product purification.
Third, some times it can happen the sequencing company or center lost the DNA, it happen to me.
Hi,
First, why you try in two different temperature? Annealing temperature is depend on the primer(s) that you designed, it not necessary to get two bands for two different temperatures. Normally we do the gradient PCR for PCR optimization. If you got band at 55, then proceed with this temperature. You can see the different when your run the electrophoresis, one is brighter and another is less brighter from PCR products from two different temperature.
Second, PCR purification, when your clean up your PCR product, make sure the concentration of PCR products are sufficient for sequencing, some times the PCR product purification can lost some or all your DNA. My suggestion is run many tubes, then elute into single tube (same temperature into same tube), so you can increase the concentration of your DNA. At the same time, how you purified the DNA, so far when I use kit no problem for PCR product purification.
Third, some times it can happen the sequencing company or center lost the DNA, it happen to me.
Optimise annealing temp through gradient PCR than run rxn on it.. U even need not to purify as most seq. Companies do it by them selves for minimum cost.
Sequencing can fail for many reasons, but the most common are:
You didn't send enough template molecules.
Your clean-up step wasn't efficient.
Did you have bright, distinct bands for all the reactions you sent in? How did you clean up the reactions?
Katie A Burnette Thanks for the reply.
I had pretty clear bands. I used the Monarch PCR & DNA Cleanup Kit.
I also took nanodrop readings to make sure the concentrations were adequate.
I think your probably right in that I didn't send enough template.
Lesson learnt.
Thanks.
You have to be very careful with nanodrop readings to zero the machine using exactly the same buffer used to elute the dna off the column, If this is TE then blank with TE not water. One other thing that can effect loading is salt concentration...too much salt means no dna loads on to the sequencing column but I think Katie is right and it is the amount of dna that is the likely problem although too little sequencing primer will also give a weak signal
if you can attach one of the .abi sequencing original sequence files for the 59c failed sequence it might help clarify the problem Sachin
Paul Rutland This is one of the .abi and the chromatogram image.
Thank you for the file Sachin,,
There are some interesting features but nothing definitive. I have great respect for MWG and have used their services for many years so it is not surprising to find the matrix is in date and all run conditions are good. The signal strength is 37 ( expect 200-4000) and the background is 6 so the signal/noise ratio is awful and the sequence unreadable but there is no sudden cut off so the problem is no DNA on the column. If this were one sample only then this could be a blocked capillary but since all of your samples show this effect then the likely causes are too little dna sequenced or the primer is either at too low a concentration or it is partially degraded ( often the case with primers dissolved long term in water not TE). This can happen as the nanodrop measures all nucleotides not just intact oligos. I think that in pcr the primer is in huge excess so some degradation is fine and will give a product but in sequencing the primer is in less of an excess so degradation makes a bigger difference to the final result.
The blue/multicolour peaks are probably capillary coating coming loose or micro air bubbles and if your sequencing was generally good I would ask MWG to repeat the sample free of charge and it would work well but as the sequencing is poor this is not worthwhile.. I have never seen signal pull up quite like your 2 plateaux and really do not know what is happening here.
Best wishes
Paul
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