disappearing bases - (Aug/10/2014 )
Hallo,
I am cloning an oligo in a vector. I just received the sequences and I noticed that almost the entire oligo was cloned in the vector. However I noticed something weird: a few of the bases were missing!
Is this something normal?
What I got was this:
Vector ..... ... oligo .... - 2bases from the oligo - 5 bases from the vector ... vector..
So on the 3' end I am missing 2 bases from the oligo and 5 from the vector. This is , in my opinion, rather weird. Or is this something to be expected?
I did not cut with a RE that would cut in those 7 bases!
So in the end, for me its ok because the oligo was just a MCS and I only lost 2 restriction sites (1 from the oligo and 1 from the vector itself) but it seems weird that this happens!
I take it that you sequenced in both directions and the sequencing primers were 30+ bp away from the site in question?
It could be either a sequencing error, or a manufacturing error.
bob1 on Sun Aug 10 20:57:13 2014 said:
I take it that you sequenced in both directions and the sequencing primers were 30+ bp away from the site in question?
It could be either a sequencing error, or a manufacturing error.
I sequenced in 1 direction, but there are many bases left at the right site (I sequenced from "left to right")
and yes, the primer was more than 30 bps from the site in question.
Its really weird: all the other sequences (left and right from the problem area) are correct! Its just that 2 restriction sites are gone!
The manufacturing error: ok, I can see this for the oligo part, where a piece is missing, but where did the restriction site from the vector go to? thats just weird!
ANd I saw it with 2 clones I had sequenced: both give the same result (the 2 sites missing!)
Star activity? I must admit, it does sound weird, I don't see how it would happen apart from there being more than one site for the RE - perhaps a mutation in the plasmid somehow?
bob1 on Mon Aug 11 08:12:51 2014 said:
Star activity? I must admit, it does sound weird, I don't see how it would happen apart from there being more than one site for the RE - perhaps a mutation in the plasmid somehow?
I have no idea what is happening.
The restriction enzyme I used to digest the unreacted vectors does not cut in the vector itself at other places.
ANd its just weird that the oligo is inserted for 90% but that the last 4 bases are not there and that 5 bases from the vector are missing, makes no sense.