First of all, I fucking HATE dealing with sequencing. It's tedious, it's boring and it takes time away from actual bench work.
At my old institution all the sequencing was done at a core facility and rarely did I get anything back that wasn't pristine. There is not core facility at my new institution, so I am using a new company and I am NOT impressed.
Sure, it could be the plasmids I sent them, but I seriously doubt it. Here's why:
I checked the concentration and purity before sending the plasmids off and they were all very good.
I am using vector-specific primers that are provided by the sequencing facility and I get the following results:
Primer 1:
gives sequence results ranging from beautiful to tolerable.
Primer 2: (same template as primer 1)
gives sequence ranging from 300 nt reads (300?!?!?) to 800 N's with a few random A's T's C's and G's dispersed throughout.
Good and bad sequence from each primer do not correlate with the template. If the worst sequence from each primer came from the same template, then I would assume the template that was the problem, but considering it did not correlate...
I'm taking a break before I blow a gasket.
Posts
8 comments:
MCLAB
Two questions: what vector aare you using, and do you have any homopolymeric tracts towards the beginning of your sequence in the reverse stretch?
The vector is pQE30 (N-terminal His tag, IPTG inducible promoter).
I don't see any homopolymeric tracts in the sequence.
I am just now getting back to it :) I'll probably be ready to explode again in about 15m.
You could always stick to animal work... =)
We've used Mass Gen's sequencing services before and been very happy with it. They're fast and relatively cheap. Good luck!
TR - LOL! Ok, so there are worse things than shitty sequence.
MH - Thanks for the tip!
Hmmmm... Tell the people in that company your Tms and GC content for the primers. They may use a std protocol ignoring any unique aspects of your vector/primers. People who do this are kind of assembly line workers; don't care much. Other option another company. We out here in LA use a local one LARAGEN and they're better than our core.
Dimitris - Excellent suggestion except that the primers were their own. Granted, that doesn't mean they know anything about them since as you said, these can be assembly line-like situations. :)
I think I'll give them one more chance, but only because I can leave my things that need sequencing at the front desk and they will pick them up.
Post a Comment