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Double digestion with BamHI and BglII - (Jul/01/2013 )

hi guys,

I need your help.
I am trying to study a putative promoter in rice and therefore I intend to use pCAMBIA1305.1 plasmid.
I will replace the 35S promoter from the plasmid and replace it with my putative promoter.

I've tried to use 2X Tango buffer as recommended by online tools from Fermentas.
I used both enzymes from Fermentas.
However, I never get any good result.

Can anyone give any suggestion?

Best regards
Apriadi

-Apriadi-

How are you telling if you get good results?

-phage434-

I've done a double digest with BamHI and BglII using NEB buffer 3.1 100X overnight at 37C. It worked out well for me - however according to my PI due to STAR activity it is better to perform a single digest with BamHI for 3hours at 37C, then continue to digest with BglII for 3 hours at 37C increasing the volume / buffers / water appropriately. 

-watsonkatherine-

https://www.neb.com/tools-and-resources/interactive-tools/double-digest-finder?enzyme1={4EC4ABBB-EF3C-4582-ABD3-367A0DA68748}&enzyme2={1A252D75-8A90-4D8C-96F6-96DB85E84CA0}

 

Double Digest Finder

https://www.neb.com/tools-and-resources/interactive-tools/double-digest-finder

-memari-

Are you aware that BglII and BamHI produce compatible ends? So if you digest your vector with these two enzymes, the vector can self-ligate without taking up an insert and it will do so much more frequently than desired.

 

If you don't use too much enzyme, combining them in buffer 3.1 should not give you any problem.

-dpo-