Entering edit mode
10.2 years ago
Fman
•
0
Dear all,
I am trying to sequence a plasmid, but I get some weird results and it seems that a region has been duplicated.
Now I am wondering: what approach might be the best in order to try and sequence in completely? Or is it pointless?
Now I have 2 contigs and by using primer walking I tried to extent the contigs in order to link them, but what happens is that rather than extending it, the new sequences are just "placed" inside the other contig or they are added to the other side of the contig. It does not extent the contig!
It is pretty weird.
any insights?
Its sanger sequencing, 1 plasmid.
Not sure I understand you. You would remove the part with the duplication and just sequence this?
The duplication is (as far as I can trust the results so far) 373 basepairs, but I think its bigger, I have to check it more.. There is something really weird going on with the plasmid.
what evidence do you have of a duplication? Just the sanger reads? When you do a restriction digest, and compare it to the parental plasmid, you should be able to figure out how much bigger it is. Through the use of a couple different enzymes, you will be able to zoom in on the region where the extra DNA is inserted. Once you know this it should be really easy to design primers and sequence the insert....
I see your point. But this will mean a lot of work! I also do not know we have the needed restriction enzymes to cut it up.
BTW: this is the parental plasmid! Its just a plasmid that I want to use, so its the parental plasmid itself ... I have not inserted anything...
And yes: the only evidence I have are the sanger reads that I tried to align to eachother using programs... I am not an expert on this type of work so perhaps I am making mistakes (I am just using the programs to align the reads, thats it and now I am trying to figure out whats going wrong).
it is not that much work. Digestion + gel will take 10' hands on work. You went through the effort of cloning and sequencing...
Yeah, thats true, I might concider it... But in short: I need to digest it and then have the digested part sequenced? Or ?
However, if the fragment is really only 400bp, there should be plenty of DNA flanking the duplication. This should allow you to figure it out?
But to be honest: the fragment with the duplication in it.. I am pretty sure I already sequenced it (not just the duplication, but much more, further parts).. with long runs! I already sequenced with many many primers (30 or something) so one should think it should already be enough to link the parts...
Its just a very very weird result!
I think you are counting on these programs too much (I know this is a bioninformatics site, but you need to understand what they are doing). Where did you get this plasmid? Do you have the sequence prior to the insertion?
I am not a bioinformaticus expert so yeah, I do use those programs, but I also looked at the contigs and checked whether they make sense and tried to make a more correct config out if it, but it does not make sense.
Its just a plasmid we have. There is no sequence before the insertion. All I have is an old sequence (that seems to be incorrect anyway) and some general information.
After looking into the sequences and leaving some out to make a more correct alignment.. still nothing! The sequence makes no sense.
I can have a look at your sequences if you want to...shoot me an email and I can see whether I can help...
That would be nice! But how do I send you an email? I can not seem to figure it out... Perhaps its because I am a new member that I can not see your email?
Hallon
how can I send you the sequence files?