help for ligation

Dr. Duncan Clark duncan at genesys.demon.co.uk
Wed Sep 25 04:15:43 EST 1996


In article <51upc9$335 at news.iastate.edu>, Qunfeng Dong
<qfdong at iastate.edu> writes
>
>Hi, sorry if this topic has been discussed before. One of my friend asked me
>what's best ratio between insert and vector. His insert is about 900 bps and
>his vector is about 12 kb, Both are cut by EcorI. Anyone experienced with
>large vector ligation before? and what's the DNA amount in ligation?
>
>Thanks a lot.
>

It should be a worked out as pM ends. For a 3kb plasmid and 3kb insert
it is usual to have a 3:1 excess of the insert ie 1ug plasmid 3ug insert
So for 1ug of a 12kb plasmid use 3ug of a 12kb insert or 0.9/12 x 3ug =
0.225ug of a 900bp insert. 

Duncan  
-----------------------------------------------------------------------------
My mind's made up. Don't confuse me with the facts!
Duncan Clark
GeneSys Ltd.




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