Scale up of ligation reaction.

Hiranya S. Roychowdhury hroychow at nmsu.edu
Thu May 18 13:54:27 EST 2000


With the right conditions you can scale up ligation reactions to 50uL. I
have had good success in that volume using the 5x ligation buffer containing
25% PEG8000. Indeed, while trying to clone some larger genomic fragments,
that was the volume that seemed to work best for me. Also, the i:v ratio
needs to be kept at 1:3 to 1:5 with larger vector sizes. If you have access
to the Sambrook et al. manual, go through the chapter therein on cosmid
cloning. You will get a very comprehensive idea about the conditions required.

At 01:14 PM 5/18/00 -0500, Rodney Earl Pettway wrote:
>I was wondering if it is possible to scale up a cosmid/genomic DNA
>ligation reaction or is it better to make several small reactions.
>I know that I post a lot of questions to this news group but I am trying
>a lot of new things and I want to get the advice of the experts before I
>try it myself.
>
>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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