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Sequencing plasmid gets overlapping peaks - troubleshoot (Aug/01/2005 )

After cloning into pGEM-T Easy, validation of the cloning was promising , but the sequencing result was too bad.. All the peaks were overlapped???? what are the reasons for that result? is it a problem in the cloning , or the sequencing ?


Could be several things... heterogeneous plasmid, impure plasmid prep, bad primer, bad sequencing. Difficult to say without looking at the electropherogram.



I am having a similar problem with my sequencing. However, in my case I have mini-preped DNA (using Promega kit), following site-directed-mutagenesis. In my case, I don't suspect the primers of being the problem, or heterogenous plasmid (since I carefully pick up individual colonies). Also, I don't think its the sequencing facility, since this is the second service I'm trying and they are giving me the same unsatisfactory output.
I was wondering if the mini-kit could be poorly isolating the DNA with some sort of contamination. If so, I was wondering if an ethanol wash, or something of that nature would be beneficial. (?) DNA concentrations seem to be very high. Also, worth noting is that this seems to be construct specific problem, since other constructs I have got sequenced have a higher sequencing success rate.

This is becoming extremely frustrating, since its is holding back my PhD project! sad.gif

Thanks for any help anyone can provide.


Hi malkhaz,
For pGEM-T easy vector, I found T7 primer is better than SP6, since sometime for the same plasmid T7 gives me good result while SP6 is bad. If your insert is not longer than 800bp you can just try T7 primer only for sequencing.

Also, can you be sure that your sequencing reagent (e.g. bigdye) is OK? But since you said "overlapping peaks", I would like to rule out this pobability. (If bigdye spoiled you will get very very low signal)


[quote=malkhaz,Aug 1 2005, 11:57 AM]
Hi, I did the sequencing last month. when I used plates for miniprep, sometimes I contaminated some templates with each other and the signal were overlap. if I extracted them individually, I got good result.


DNA sequencing is something I unfortunately know too much about wink.gif

Many things can cause overlapping or mixed peaks:

1. Double picks or double colonies
2. PCR occurring in the sequencing reaction. This results from the primer binding down stream of the correct site such that you get a PCR product with the primer binding site on each end.
3. Dirty DNA contaminated with RNA, proteins, and genomic DNA
4. Failed reactions. In this case the sequencing software pulls up the machine noise and makes it look like mixed signal.
5. Primer binding to a secondary site on the template.
6. Two primers. You would be amazed at the number of people who manage to add two primers to their sequencing reactions.
7. Cross contaminating your plasmid DNA during the miniprep process.

As you can see it is hard to give advice without knowing more of what you have done.


DNA sequencing reagents

-Daniel Tillett-