Help!!!!!! Ligation

[John Brennand]

Here's my twopenneth

It is easy to check whether your ligations are working - set up 

vector alone
insert DNA alone
vector + control PstI fragment
vector + insert DNA (vary the ratios, 1:1, 1:3, 1:10)
All +/- ligase overnight.  

Run 1/3 of the DNA's on a gel and see whether the patterns are as 
expected.  If they are not, I would suspect that the long PstI 
incubation has "nibbled" the ends - use more enzyme for less time.
If they look OK - transform.  If you dont see any colonies and the 
transformation control is good I would suspect that the insert DNA 
is being restricted by residual enzymes in the host strain - 
therefore try alternative host strains to transform.

John