ligation reaction problems!!

Rodney Earl Pettway earlpettway at netscape.net
Fri May 12 17:31:22 EST 2000


Thank you very much.  That just made my life a lot easier.

"Hiranya S. Roychowdhury" wrote:

> If you are making a library, you use the avearge fragment size to determine
> that.
> 1pmol of a 1kb DNA is roughly 660ng
>
> the rest is elementary physical chemistry.
> (1mole/liter = 1M)
>
> For your situation (ie. 9:1), say your cosmid is 10kb; you need 6.6ug of the
> cosmid to get 1pmole, OR, 9x6.6ug =  59.4ug to get 9pmole
>
> If your average insert size is 20kb, you need 20x660ng =  13.2ug to get
> 1pmole of insert.
>
> Hence, for every rxn you set up, you should have the above amounts of your
> cosmid and genomic DNA.
> Now, nobody uses that much, unless you want to do a library. Even then you
> should probably use in the ng range and set up several ligations.
> For a single insert, I would use about 5.9ng of the vector and 1 to 1.3ng
> insert.
>
> Remember, the above is just an example.
>
> At 03:55 PM 5/12/00 -0500, Rodney Earl Pettway wrote:
> >I would like a detailed protocol for the ligation of cosmid DNA with
> >genomic DNA.
> >I have one but it ask me to set up a reaction containing a 9:1 molar
> >ratio of  vector DNA:eukaryotic DNA.  I don't know how to figure out the
> >molar concentrations.
> >
> >thanks
> >
> >Rodney Earl Pettway
> >USDA-ARS
> >Cereal Disease Lab
> >University of Minnesota
> >
> >
> >
> >
>
> Dr. Hiranya Sankar Roychowdhury
> GENE LAB/ EPPWS
> New Mexico State University
> Las Cruces, NM 88003
> Ph. (505) 646-5785
> hroychow at nmsu.edu
>
> ---





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