problem in X-gal plate - (Nov/06/2008 )
Hi,
Can anybody find a solution to my problem 
I am cloning a 400bp promoter in 11 kb plasmid which is LacZ minus. when i transform the intact plasmid i got white colonies on the X-gal plate. When I went ahead with digestion, ligation and transformation I am getting blue colonies in self ligation!!! sometimes there is a bluish tinge even with uncut vector on X-gal plate. I am using 30ug/ml concentration of the X-gal. Is it very high? I want to get rid of blue colonies from self ligation and uncut vector. I changed the water and buffer but still the problem persists. Please tell me a way out
-Abhay Bhat-
QUOTE (Abhay Bhat @ Nov 6 2008, 08:41 PM)
Hi,
Can anybody find a solution to my problem I am cloning a 400bp promoter in 11 kb plasmid which is LacZ minus. when i transform the intact plasmid i got white colonies on the X-gal plate. When I went ahead with digestion, ligation and transformation I am getting blue colonies in self ligation!!! sometimes there is a bluish tinge even with uncut vector on X-gal plate. I am using 30ug/ml concentration of the X-gal. Is it very high? I want to get rid of blue colonies from self ligation and uncut vector. I changed the water and buffer but still the problem persists. Please tell me a way out
Can anybody find a solution to my problem
I am cloning a 400bp promoter in 11 kb plasmid which is LacZ minus. when i transform the intact plasmid i got white colonies on the X-gal plate. When I went ahead with digestion, ligation and transformation I am getting blue colonies in self ligation!!! sometimes there is a bluish tinge even with uncut vector on X-gal plate. I am using 30ug/ml concentration of the X-gal. Is it very high? I want to get rid of blue colonies from self ligation and uncut vector. I changed the water and buffer but still the problem persists. Please tell me a way outIf u transform intact plasmid u got white colonies..
then when u claimed self ligation u got blue colonies
intact plasmid = self ligated plasmid?! assuming that ur restriction site is unique this can't be right.
more info needed
-Hanming86-
QUOTE (Hanming86 @ Nov 9 2008, 05:14 AM)
QUOTE (Abhay Bhat @ Nov 6 2008, 08:41 PM)
Hi,
Can anybody find a solution to my problem I am cloning a 400bp promoter in 11 kb plasmid which is LacZ minus. when i transform the intact plasmid i got white colonies on the X-gal plate. When I went ahead with digestion, ligation and transformation I am getting blue colonies in self ligation!!! sometimes there is a bluish tinge even with uncut vector on X-gal plate. I am using 30ug/ml concentration of the X-gal. Is it very high? I want to get rid of blue colonies from self ligation and uncut vector. I changed the water and buffer but still the problem persists. Please tell me a way out
Can anybody find a solution to my problem
I am cloning a 400bp promoter in 11 kb plasmid which is LacZ minus. when i transform the intact plasmid i got white colonies on the X-gal plate. When I went ahead with digestion, ligation and transformation I am getting blue colonies in self ligation!!! sometimes there is a bluish tinge even with uncut vector on X-gal plate. I am using 30ug/ml concentration of the X-gal. Is it very high? I want to get rid of blue colonies from self ligation and uncut vector. I changed the water and buffer but still the problem persists. Please tell me a way outIf u transform intact plasmid u got white colonies..
then when u claimed self ligation u got blue colonies
intact plasmid = self ligated plasmid?! assuming that ur restriction site is unique this can't be right.
more info needed
Hi,
first of all when I transform uncut plasmid itself I am getting very slight bluish color to those colonies. The plasmid suppose to show jet white colonies coz it doesnt have promoter. am cloning a promoter in EcoRI-BamHI sites which are unique and in the self ligation I am getting 2-3 jet blue colonies and a couple of white colonies! Its a mixture.... but am not getting colonies properly in the test sample(vector+insert). How can it be possible?? I am getting blue and white colonies on self ligation and no colonies in the test sample!!! am really bugged up..
All suggestions are welcome...
-Abhay Bhat-