ligase three fragments - ligase three fragments (Nov/08/2002 )
How to ligase three fragments? The vector is 5.5kb.The insert fragments are 5kb and 6kb.The vector are digested by EcoRI .And the two fragments are digested by EcoRI and BamHI.I have done serveral times But only get serveral self-ligating vector.How to do ?
I don't know how you got your fragments. One thing you should keep in mind is that the restriction site should not be too close to the ends of your fragments (if you are not cutting your fragments directly from another vector).
May sound trivial, but have you tried dephosphorilating the vector after digestion.
And the other thing is, I would consider a step by step approach, i.e. ligate one insert + vector, tranform it into E.coli, reisolate the rest, digest it, go for the next insert.
Never give up, never surrender!
Dephosphorylate the vector with Shrimp Alkaline phosphatase (rather than the more finicky BAP or CIP). This enzyme can be used in the EcoR I digest so that the vector is simultaneously cut and dephosphorylated. Heat inactivate both enzymes at once.
Given their nearly identical sizes, use a 1:1:1 ratio for the three fragments.
Normally with a three fragment ligation, you can expect a 10-fold reduction in colonies when compared with a two fragment ligation.
The final size of your plasmid is rather large at 16.5 kb. Normal pipette tips can shear this DNA during handling. Use wide bore tips or cut off the ends of your current tips with a clean razor blade.
Stability may be an issue. A cosmid vector may be more suitable. Also, there are E. coli strains designed for large plasmids available commercially.