ligase three fragments
Posted 08 November 2002 - 11:51 PM
Posted 09 November 2002 - 03:10 PM
Posted 12 November 2002 - 02:30 AM
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!
Posted 20 November 2002 - 03:29 PM
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.