3 piece ligation - (Jun/15/2008 )
Hello
Anybody has done 3 piece ligation in the past successfully. I am trying to join a three piece of DNA having two Hpa I site and one XmaI site. After ligation i am getting the ligation product in the form of a ladder which i suppose is a multiple combination of DNA fragments in the linear form and not the supercoil. I am not getting any colony after transformation. Please give me the conditions if anyone knows it how to do that.
sunil[/b][/b]
-sktewary-
QUOTE (sktewary @ Jun 16 2008, 08:46 AM)
Hello
Anybody has done 3 piece ligation in the past successfully. I am trying to join a three piece of DNA having two Hpa I site and one XmaI site. After ligation i am getting the ligation product in the form of a ladder which i suppose is a multiple combination of DNA fragments in the linear form and not the supercoil. I am not getting any colony after transformation. Please give me the conditions if anyone knows it how to do that.
sunil[/b][/b]
Anybody has done 3 piece ligation in the past successfully. I am trying to join a three piece of DNA having two Hpa I site and one XmaI site. After ligation i am getting the ligation product in the form of a ladder which i suppose is a multiple combination of DNA fragments in the linear form and not the supercoil. I am not getting any colony after transformation. Please give me the conditions if anyone knows it how to do that.
sunil[/b][/b]
stop yelling and check this post and if you can, do multiple step cloning.
-ph3no-
QUOTE (ph3no @ Jul 7 2008, 01:59 AM)
QUOTE (sktewary @ Jun 16 2008, 08:46 AM)
Hello
Anybody has done 3 piece ligation in the past successfully. I am trying to join a three piece of DNA having two Hpa I site and one XmaI site. After ligation i am getting the ligation product in the form of a ladder which i suppose is a multiple combination of DNA fragments in the linear form and not the supercoil. I am not getting any colony after transformation. Please give me the conditions if anyone knows it how to do that.
sunil[/b][/b]
Anybody has done 3 piece ligation in the past successfully. I am trying to join a three piece of DNA having two Hpa I site and one XmaI site. After ligation i am getting the ligation product in the form of a ladder which i suppose is a multiple combination of DNA fragments in the linear form and not the supercoil. I am not getting any colony after transformation. Please give me the conditions if anyone knows it how to do that.
sunil[/b][/b]
stop yelling and check this post and if you can, do multiple step cloning.
Yellers never make 3 piece ligations to work, so 's the point?
Tell us if it worked, anyways!
-cellcounter-