Ligation of GFP(LVA), pBR322, and HindIII-EcoRI Adapters

 

Partly from Sambrook, 1.68.

Materials:

Methods:

1. Make a more concentrated solution of DNA fragments pBR322 and GFP(LVA), by using total of the pBR322 vector and GFP(LVA) insert with a microcon centrifugal filtration device (according to Protocol G). Result: of solution. DNA fragments should be in TE (pH 7.6).
2. set up tubes to anneal and ligate adapters, GFP(LVA), and pBR322 vector:

   

3. Incubate solution for 5 min to melt any cohesive termini that have reannealed. Chill the mixture to .
4. add the following to tube (for a total of ):

   

5. Incubate the reactions for 4-16 hours (rather than suggested 1-4 hours ).

Results: ( 2/1/99)

First, attempted to get a more concetrated form of the DNA fragments using cetrifugal filtration. The first filtering step seemed to work fine. However, we could not get the DNA out with 3 min of 1,000 g. Therefore, ended up using 30 sec of 14,000 g, which got us about . Finally, mixed the solutions (with ligase and ligase buffer by accident), warmed to for 5 min, added ligase and ligase buffer, and put .