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Huge problem in cloning, need help quite urgent - (Feb/01/2006 )

Hello every one,

I have a big problem, is already 2 months I'm trying to clone a plasmid, basically I'm linearizing my vector (AvrII site, size=7kb) and I'm planning to introduce a XbaI insert (700 bps).
It seems quite simple but until now either I have not got colonies or I'm getting completly wrong plasmids.
I would like to know the amount of DNA you are using for this kind of ligation (I have read some where that 25 to 50 ng of vector is enough).
I hope some one could help me...

best wishes
Marco Antonio

-marco-

QUOTE (marco @ Feb 1 2006, 06:03 PM)
Hello every one,

I have a big problem, is already 2 months I'm trying to clone a plasmid, basically I'm linearizing my vector (AvrII site, size=7kb) and I'm planning to introduce a XbaI insert (700 bps).
It seems quite simple but until now either I have not got colonies or I'm getting completly wrong plasmids.
I would like to know the amount of DNA you are using for this kind of ligation (I have read some where that 25 to 50 ng of vector is enough).
I hope some one could help me...

best wishes
Marco Antonio


sorry but i didnt understand why do you linearize your vector? yes i used 50ng of vector with a ratio of 1:3 vector/insert and had great results....ratios can vary but 50ng of vector is definitly enough i think....but since you are using single digestion then I think your vector is religating....(ligating to itself once more) you have to treat it after digestion....sorry cant remember with what ....im sure others will help more.....are you cutting your insert from another vector? or is it PCR product?

-Kathy-

Hi Marco,

Kathy is right, the most favourable ligation product will be re-ligated vector. When I do blunt end ligations I commonly use approximately 100ng of DNA but 50 should be fine and I tend to try and drive the reaction by using vector/insert ratios of between 1:3 to 1:10. Given that you are only getting artifact or nothing I would bump the concentration of DNA up in the reaction even up to 200ng of vector.

Also as Kathy suggested always de-phosphorylate your vector with Calf Intestinal Alkaline Phosphatase (CIP) this will reduce your background.

As you are cutting your insert with XbaI you will not have to phoshporylate your insert ends.

Another thing is the insert from vector or PCR as you will have to make sure the XbaI is cutting the PCR product if applicable, as restriction enzymes are not very efficient near the ends of PCR products.

Do you get lots of colonies on your control plates, to check for transformation effeciency?? Is anyone else in the lab having problems ligating, I went through a stage I couldn't ligate anything, turned out to be contaminated water.

Hope this helps

Scott

-Scott-