My problem: sequencing result is low-quaility and dirty. Most sequence is matched with expected one, but there are some sites having two bases' signal, and the overall base signal is not strong, but around 60th bp position, there is a very high peak.
My rough experiment flow: purified plasmid DNA, Big Dye v3.1 protocol to do sequencing PCR, FastGene Dye terminal removal kit, then sequence in machine (using Medium-seq).
Primer is 100 bp ahead of my target region, primer annealing region is a fragment of GFP protein.
In fact I have tried DNA amount of 100ng, 200ng, 500ng, 1000ng, but they all did not have clear result. Only in 500ng, only one of the sample showed a bit good result and matched with expected result, but other samples were just so-so or bad whatever the DNA amount is.
Please help me! I can give more information if needed.
It looks like you are using too much dna but that the real problem is the amount of primer used in the sequencing reaction, How long is your pcr product and how much primer are you using in the sequencing reaction and is this a newly purchased primer or could it be starting to degrade. Have you tried sequencing using the revebse primer ?
Paul Rutland , Thank you so much for your answer.
I add 5 pmol primer for the sequncing PCR for all the reactions I tried. Is it a problem? Should I change the amount according to DNA amount? The product usually is 600bp.
In fact I have tried 3 primers, results are no difference. two primers (forward and reverse) is newly purchased, one forward primer is from my lab mates he verified before. But the time it stored is not long I think.
By the way, after plasmid purification, I measured them in NanoDrop, the A260/A280 is around 2.0,,, will it influence quality?
The amount of dna template is given by 25ng x (length of template in KB) so if your dna template is 600 bases then the amount to sequence is 25 x 0.6 or about 15 ng If the 600 bases were in a plasmid then the amount would have been 25 x 600 plus the length of the plasmid in kb so I think that you are using too much template. Unfortunately the od260/280 is greater than 1.8 so you may have some rna or salt contamination making it a bit difficult to calculate exctly how much dna you are adding. If you are sequencing with plasmid primers for an inserted template then liearising the plasmid may help The very high peak at 60bases shows that the sequencing reaction has not worked and the purification process after the sequencing reaction has not worked completely so this peak suggests that either the sequencing kit is failing ( has any sequencing worked) or that the primers did not bind well ( lower the annealing temperature) It would be intersesting to see an original .abi sequence file which may carry more information for troubleshooting
It seems that you are using to much amount of DNA. Basically 20 ng as a total mass is sufficient for DNA length 1-1000 bp. High DNA concentration will produce overlapping chromatogram picks.
Ammar Elakhdar .Thank you so much for your answer. I will take your advice carefully.
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