Scale up of ligation reaction.
Rodney Earl Pettway
earlpettway at netscape.net
Thu May 18 13:51:05 EST 2000
Thanks again for your help!!!!!!!!!!!
Have a great day!
"Hiranya S. Roychowdhury" wrote:
> 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.
> >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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