In total, I have a gene which has 896 bases. After DNA sequencing process (as I mentioned in the title), approximately both are on the beginning portion. Almost 100 bases at the beginning portion, and the ending portions have almost 100 bases. These appear to be like mixture, two or three pick seemed in the one base section of my gene. But for the remaining part in the middle, about 400-500 bases have a completely clear dna sequence all the time.
What is going wrong in the beginning and ending sections? Truly, I wonder it.
I would improve the purification step which can be the reason of poor quality of your sequence. Plus use the right amount of DNA suggested by the manufacturer.
It is normal to have at the end of your sequence a decrease in quality especially if you are sequencing long fragments. To read completely your sequence use a couple of forward primers, just to be sure to cover the entire region.
What method do you use to purify your sequencing reaction?
Let me know and good luck,
E
This is normal when you use sanger sequensing. It is because at the beginning of the sequencing run, some nucleotides fluoresce more than others & mask the other peaks, while at the end of the sequencing run, the fluorescence is too weak & the peaks overlap each other. To overcome this, you can either use primers outside the region of the gene of interest, or use along with the forward & reverse primers you are using now, 2 internal primers that start at the middle of the gene & run to the beginning & the end of your gene of interest. The second option is better since you'll have at least 2x coverage of each nucleotide, however, it is also twice as expensive.
This is normal, using Sanger sequencing. To overcome this, you can cloned the PCR product and than sequencing.
good luck
Thanks for all assistances. Dear Naji, I examined all written. I understand how I use different primers and why. Thank you.
So Dear Emanuele, we use after first PCR, Exosap purification. After that, we do Pcr once again. So we use Sanger sequencing. So suggestions which is about primers and their sections on gene, thank you particularly.
So Dear Krishna, we have a lot of sample about same gene. So we cannot deal with the cloning. If we have a small amount sample, we'd do that. Thank you :)
So Dear Arthur, why do you think noise which at the beginning section of gene about 100 bp isn't normal? So how many bp is normal at the beginning both at the ending about noise ?
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