June 28th, 2007

Fizzgig

This Ligation is killing me....

Hey everyone.

Molecular biology post/rant follows. Feel free to ignore if you're not interested....

I have been stuck trying to get this one ligation to work, and it is killing me....

I have a 3.3kb insert, and a 2.6kb vector. It's not like they're both over 6kb or there's a 15 kb insert or anything. Should be child's play!

Vector is getting cut with XhoI and XbaI, and then CIP'ed, 2 hour digest of each (NEB enzymes and buffer#2). Insert is getting digested out of a different plasmid, same enzymes and buffers as above, but no CIP.

Both are run on 0.5x TAE gels, band are cut, and then cleaned up via Qiagen spin columns....

ODs are taken, and I have set up ligations ranging from 1:10 to 10:1 i:v ratios.... (usually ~50 ng vector).

I have tried this multiple times with each of multiple preparations of the friggin insert and vector, and nothing is working!!! arghhh!!!!!

Anyone have suggestions? For size ratios like those, what i:v ratio do you guys normally use?

I'm dying here.....

Matt
  • Current Mood
    frustrated frustrated
Science!

The care and feeding of vacuum lines.

Dear world: The vacuum lines are not some magical disposal system. Sucking up litres of fluid into it does not send it to the disposal fairies, where it frolics in its happy, EPA approved disposal category.

What it does, instead, is floods the whole bloody vacuum system for the floor with mysterious blue substance that leaks out various spigots like a fountain of unknown waste. Further, it shuts down every project on said floor that requires use of said vacuum lines. God only knows what it does to the vacuum pump.

In conclusion, use a bloody fluid trap, or we will ruthlessly hunt you down for bring people's research to a crashing halt.
  • Current Mood
    enraged enraged