I need help on Ligations

Ian Mehr ijmehr at merle.acns.nwu.edu
Tue Apr 18 11:19:29 EST 1995


In article <3mef05$4ct at Twain.MO.NET>, akravetz at MO.NET (Andy Kravetz) says:
>
>Okay, I pouring out my heart and soul because I can't get the !@#$$ thing 
>to work. 


Andy,

I hear your plight.  For the last year, I have had troubble with ligations. 
 I have been attempting to ligate 5-7kb random fragments of a bacteria's
 genome into a 2.3kb vector to create a genomic library.  You may guess that
my attempts have been frustrating.  One thing you did not address
 in your post was wether you phosphatase your vector or not to prevent its religation.
The absence of transformants in your vector control is disconcerting to say the least.
How do you transform?  This may be your problem.  Furthermore, changing from molar to 
mass ammounts in your ratios most likely won't make a difference (at least none that
I could logically think of - and I tried it as well).  Also, any text I've read has stated
that beyond a 1:2 ratio is a waste of material, as your effeciency of ligaiton of vector to
insert does not concomittantly increase.  It would most likely be better to vary the
concentration of your ligation mix should you want to play with any variables.

With out taking up to much space, thats all I can get into. Let me know if you have any luck.


Ian J. Mehr
ijmehr at merle.acns.nwu.edu



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