karthi at deakin.edu.au (Karthi Ramachandran) wrote:
>In article <199610161736.MAA06281 at wustl.edu>, brett at BORCIM.WUSTL.EDU (brett) wrote:
>>>I'm having difficulty cloning a 7 kb Sac I fragment into pUC 19. One
>>>problem has been the phosphotasing of pUC 19/SacI. I phosphatased the
>>>vector twice with shrimp alkaline phosphotase. Comparing transformations
>>>of vector without ligase and vector with ligase, I get thousands of blue
>>>colonies for the vector with ligase. Self ligation of the vector in the
>>>absence of insert has also been confirmed by gel electrophoresis.
>>>Apparently the 5' *recessed* phosphates are not being removed completely.
>>>>Yeah, those recessed phosphates can be tricky. I'd use 2-3x excess CIAP(!) at
>>50C for 1 hr, then clean up the vector. Your background should be very low.
>>>I use CIP and when phosphotasing blunt and recessed ends, I incubate the
>reaction with 1 unit of ez. at 37 for 30 min , add another unit and continue
>incubation at 55 for 1 hr. This works for me
>Karthikeyan Ramachandran Ph.D
I have used a similar protocol for phosphatase treatment of recessed
or blunt ends. It works like a charm!
To a 50 ul restriction digest of vector add 0.5 ul (0.5 Units) of CIP
(Boehringer), 0.5 ul of 1M Tris-HCl, pH 9.0 and 74 ul of water.
Incubate at 37C 15 min and at 55C 15 min. Add another 0.5 Units of
enzyme and repeat incubations. Phenol-ChCl3 and ChCl3 extract to get
rid of tenacious enzyme, followed by Ethanol precipitation. I usually
gel-purify the vector to remove undigested plasmid which can add
significantly to background cfu formation, but this step may be
omitted when you have a blue/white screen.
Julie A. Kirihara, Ph.D.
ATG Laboratories, Inc.
email: kirihara at gold.tc.umn.edu