Re: [ccp4bb] Phosphatase, which is the best?

- Protein crystallography

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CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

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Subject: Re: Biorad Profinia vs GE AKTAPrime Plus, for affinity tagged proteins
From: Chun Luo chun {- at -} ACCELAGEN {- dot -} COM
Date: 2010-01-28

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Subject: Re: Phosphatase, which is the best?
From: Chun Luo chun {- at -} ACCELAGEN {- dot -} COM
Date: 2010-01-28

NEB recommends adding Zn for Antarctic Phosphatase. Although we found it
works in all NEB restriction enzyme buffers.

The answer can be found at
http://www.neb.com/nebecomm/products/faqproductM0289.asp.

--Chun

_____

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Akram
Alian
Sent: Thursday, January 28, 2010 9:32 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Phosphatase, which is the best?

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:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Nenci
Simone
Sent: Thursday, January 28, 2010 6:16 AM
To: CCP4BB@JISCMAIL.AC.UK
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

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: Biorad Profinia vs GE AKTAPrime Plus, for affinity tagged proteins
From: Chun Luo chun {- at -} ACCELAGEN {- dot -} COM
Date: 2010-01-28

Next message:
Subject: Re: Refining against images instead of only reflections
From: John Badger jbadger1 {- at -} SAN {- dot -} RR {- dot -} COM
Date: 2010-01-28