4 replies to this topic

[deespike]

Hi all

I am unfortunately involved in a series of blunt ligation and i donot have any option other than that. I want to ligate a 2.3kb insert cut with EcoRV into a 12kb vector cut with Smai.  I use neb enzyme upto 800 cohesive ligation end units , tried 22C, 6:1 :: insert:vector ratio but i dont get anything. I didnt dephosphorylatet the vector but still no self ligated colonies are there. The comp cells are okay (checked with plasmid).

Is there anything i can do as this is very frustrating?

Anyone having sugggestion for successful ligation?

[NemomeN007]

deespike, on 23 August 2010 - 07:22 AM, said:

Hi all

I am unfortunately involved in a series of blunt ligation and i donot have any option other than that. I want to ligate a 2.3kb insert cut with EcoRV into a 12kb vector cut with Smai.  I use neb enzyme upto 800 cohesive ligation end units , tried 22C, 6:1 :: insert:vector ratio but i dont get anything. I didnt dephosphorylatet the vector but still no self ligated colonies are there. The comp cells are okay (checked with plasmid).

Is there anything i can do as this is very frustrating?

Anyone having sugggestion for successful ligation?

Are you sure you're using the correct selection plates for your vector?  Your test plasmid may be Amp, but your cloning vector is likely something else.

[deespike]

My selection agent is kanamycin . I am using the right plate.

[Clare]

Try a 16degC overnight incubation and add 2.5ul of 50% PEG 4000 (or 6000 etc) per 20ul ligation reaction. This is what I always do and my cloning always works
Good luck!
Clare

deespike, on 23 August 2010 - 07:22 AM, said:

Hi all

I am unfortunately involved in a series of blunt ligation and i donot have any option other than that. I want to ligate a 2.3kb insert cut with EcoRV into a 12kb vector cut with Smai.  I use neb enzyme upto 800 cohesive ligation end units , tried 22C, 6:1 :: insert:vector ratio but i dont get anything. I didnt dephosphorylatet the vector but still no self ligated colonies are there. The comp cells are okay (checked with plasmid).

Is there anything i can do as this is very frustrating?

Anyone having sugggestion for successful ligation?

[perneseblue]

Clare, on 23 August 2010 - 11:10 AM, said:

Try a 16degC overnight incubation and add 2.5ul of 50% PEG 4000 (or 6000 etc) per 20ul ligation reaction. This is what I always do and my cloning always works
Good luck!
Clare

deespike, on 23 August 2010 - 07:22 AM, said:

Hi all

I am unfortunately involved in a series of blunt ligation and i donot have any option other than that. I want to ligate a 2.3kb insert cut with EcoRV into a 12kb vector cut with Smai.  I use neb enzyme upto 800 cohesive ligation end units , tried 22C, 6:1 :: insert:vector ratio but i dont get anything. I didnt dephosphorylatet the vector but still no self ligated colonies are there. The comp cells are okay (checked with plasmid).

Is there anything i can do as this is very frustrating?

Anyone having sugggestion for successful ligation?

If all else fails, the alternative is to avoid blunt end cloning and revise the ligation strategy to allow for sticky end ligation or use the invitro homologous recombination kit from clontech.