ligation reaction problems!!

Hiranya S. Roychowdhury hroychow at nmsu.edu
Fri May 12 17:31:16 EST 2000


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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