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About Ligation :ph34r: - (May/31/2006 )

HI everyone...
I have tow Questions

1) I must to construct two plasmid. The first one have a vector of 6,2 kb and insert of 0,4 kb. The second one use the same vector but with insert of 1,2 kb.

Which could be the correct ratio for ligation in the two cases?

I commonly use 1:10 vector:insert.... is it to much?

2) I´m trying to do a ligation of insert with 5' NruI (blunt) and 3' NheI (sticky) with a vector wich have NruI and NheI ends. I carry out the ligation as follows:

- 200 ng of vector
- Vector:Insert in 1:5 molar ration.
- 1 unit of Promega's T4 Ligase
- The total volumen of mix is 10 uL.
- I keep the mix 3 hours/22°C and then 16hours/16°C.
- I transform competent cells with 5 uL mix.

I dont know what Im doing wrong... someone have an idea?
Thanks a lot for your help.




try Vector:Insert in 1:3 molar ration.
may be it helps you.