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No sequencing of DNA methylation - (Aug/14/2014 )

Hi, I need help...


I was able to sequencing good sequences of converted DNA, my workflow is DNA purification, converting DNA (Kit) ,  specific PCR, cloning, PCR confirmation of colonies, plasmid extraction and send the plasmid to sequencing with specific primers, my samples are in the right concentration and the size is about 300bp. But at begining of year something happend and now my sequence are how the picture show... The `A` is how my sequences are at the moment (bad), the `B` is how some sequences appear, the `C`and `D`show how then were... Good! The good sequences agreed with the reference.

I don`t known what happening, I have read about the use of SP6, I have already tried... I read about the hairpin that can form due to the excess of repeated bases in conversion... The use of more formamide...

Someone could help me??





(sorry forAttached Image bad english)

-Tati GM-

Are you sending miniprep plasmid DNA from a single colony? What primer are you using? Is it a primer binding to the vector, or to your DNA sequence? This should have little or nothing to do with the converted DNA if you are sequencing a plasmid with a vector binding primer.


Thanks for your reply.


Yes I sent a single colonie, I am trying sequencing with T7 primer because I used pgem T.

-Tati GM-

This suggests that the problems you have are unrelated to anything except sequencing of the plasmid. You should try control sequencing of a known good plasmid (doesn't matter what it is,  presumably a pGEMT plasmid). It might also be prudent to streak out your "single colony" for single colonies again, to assure that it is not a mixed population. Make sure your DNA submitted for sequencing has the correct concentration. Both too l ittle and too much DNA can cause problems in the sequencing reaction. Make sure your primer is correct, and at the correct concentration.


Thanks again,


I`ll check everthing again, I`ll found a control too, but I follow the protocol (150-300ng / DNA) I used Qubit to check, after delivering the samples to sequencing I don't have control about happens... It's worried me... But I can do the process too!?

-Tati GM-

Since you are having trouble, I would check the DNA concentration on a gel, not with Qubit. Most ladders (such as NEB 2-log ladder, my favorite) have DNA concentrations associated with each band, if you run a known amount of ladder. Compare your band with one of the ladder bands and estimate the DNA concentration. What counts is the DNA concentration of your desired plasmid, not the total DNA concentration.



The last test: I checked the concentration (I let all the samples around 280ng), I sent a control, duplicate and I used M13 primer foward and reverse. Total of samples 8, only 5 worked, same in duplicate, the result was the same in both. I still do not understand...How I could attach the picture in reply?

-Tati GM-