Dear Nenci
I always used CIP with great success. A good control to test
whether CIP worked, a plate with a plasmid (containing no insert) should not
ligate and will not be circular, hence if CIP worked, this plate should not
contain any plasmids grown on it (or very few).
However, if you were to clone a gene, a plasmid that contains the
inserted gene (which will bring in the two phosphates for the two dephosphorylated
ends) will ligate and colonies will grow.
I never tried SAP, thus cannot comment on that
Good luck
Akram Alian, Ph. D.
Department of Biochemistry & Biophysics
UCSF
600 16th street, S414
San Francisco, CA-94158
Phone: (415)-476-3937
From: CCP4 bulletin board
[mailto:[log in to unmask]] On Behalf Of Nenci Simone
Sent: Thursday, January 28, 2010 6:16 AM
To: [log in to unmask]
Subject: [ccp4bb] Phosphatase, which is the best?
I need to dephosphorilate blunt ends and, surfing on
internet I found this two enzymes:
- Shrimp Alkaline Phosphatase (SAP)
- Calf Intestinal Alkaline Phosphatase (CIP)
Have you any experience about these phosphatases, I mean wich is the best for
blunt ends?
All the best
Nenci Simone
-------------------------------------------------
Dept. Genetics and Microbiology
University of Pavia
Via Ferrata 1, 27100 Pavia
Italy
Tel. +39-0382-985535/985534
Fax +39-0382-528496
Web www.unipv.it/biocry