digestion and ligation with BspH1 - digestion with BspH1 of insert , and Nco1 for the vector ,ligation pos (Apr/15/2007 )

hi all
need help with ligation !

i am cloning GFP gene just before my gene into pRTL2 vector to get a fusion protein expression in plant.

the insert gene has to be cloned into the Nco1 site of pRTL2 vector , since it contains the start codon.
but my GFP has Nco I site in the middle , hence a problem creating NcoI site at the 5'end for pcr

i refered some papers , got one that they used BspHI ( pagI) at the 5'end and XbaI at the 3'end of this same GFP gene and cloned into NcoI and XbaI digested pRTL2 vector .

i tried this way too since that would mean i dont have to do mutagenisis which might take more time , but after transformation all colonies only contain the vector with my gene , self ligated . no GFP gene.
i had previously done GFP tagging at the 3'end of my gene , it went well . so size is not a big problem or transformation ( heat shock) into Xl1 blue.

the elution of digested prodcuts were good and

i used 1:3 and 1:5 vector :insert ratio ,o/n incubation at room temperature

is there anything that i need to look out for? like ligase or ligation conditions?

NcoI site is C/CATGG

and BspHI is T/CATGA

or does it mean that i will need to screen more colonies to check ?

is there a possibilty of working?

any suggestions welcomed

thanks in advance

laxmi

I have done similar thing, NcoI and BspHI sites at one end and XbaI at the other, 1:3 vector :insert ratio, o/n ligation at 4 C, and it worked well.
I do ligation of gel purified fragments

If you are using NcoI only don’t forget to dephosphorylate vector. Design primers with BspHi sites at both ends of GFP, and there is no need for mutagenisis.

thank you very much , Kajmak

i will try ligation at 4 degree ,
i use double digestion, the other site is KpnI so no problem with that only NcoI and BspHI

thanks a lot i was not very confident it would work .
ur reply given me hope
i did ligation and transformation twice and screened 30 colonies , i will try 4 degree instead of room temp
thanks again
laxmi