blunt-end ligation

Dr. Hiranya S. Roychowdhury hroychow at nmsu.edu
Thu Oct 26 19:03:51 EST 2000


Assuming you already have good success in sticky-ends, do the ligation at
RT in a volume of 10uL to 20uL. 
I rutinely do blunt end ligation as follows:

5ng vector (dephosphorylated)
5ng insert
2uL of the 5x ligation buffer (FOCUS; BRL buffer)
1 U ligase
total vol. to 10uL
30 min to overnight at RT

Transform comp. cells (100uL) with 5uL of the rxn.
Recover at 37 C w/ very gentle shaking for 30-45min.
Plate.

If you are not making a library of some sort, this will be just fine.

I'm in a rush. If you need a more detailed protocol, email me.

ciao

Hiranya.

At 10:16 PM 10/26/00 +0100, Rodney Earl Pettway wrote:
>Anyone have a good blunt-end ligation protocol?
>
>"PEACE"
>
>--
>ÐÏࡱá
>
>
>Attachment Converted: "c:\eudora\attach\earlpettway.vcf"
>
Dr. Hiranya Sankar Roychowdhury
College Asst. Prof.
Molecular Biology,
Dept. of Chemistry & Biochemistry	
Box 30001 - 3MLS
New Mexico State University
Las Cruces, NM 88003

Lab: (505) 646 4722
Office: (505) 646 8256
hroychow at nmsu.edu


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