Ligating large fragment into vector

[Graham Dellaire]

Hello Jimmy,


First of all my advice is to check whether your pcr primers
are phosphorylated....hehe common mistake.  If they aren't then do
a simple T4 Tk reaction and heat denature the enzyme before proceeding 
with any cloning.  

Now you mention that after cutting you still can't ligate....hmmm this 
should leave a phosphate... so perhaps your efficiency is low and the 
bugs (bacteria) you are using to clone the plasmid are really low 
efficiency and you are missing the clone...by the number game.

Also perhaps your product is toxic in your bacterial cells?   don't 
know.... need more info about your situation (what DNA what 
vector...etc.)

Even if one site isn't cutting you should get ligation anyway....if you 
really want to know you will have to plate 5- 10 plates at about 10-2 
dilution and then do colony lift....it is really easy and you can probe 
with nick translated probe from your pcr fragment.  I once cloned 16 kb 
of adeno-viral dna into a 7 kb vector...yep 23 KB plasmid...it worked 
although I had to screen 50,000 cells by colony lift.  I did this in one 
experiment and the rate of insert to false positives was 1 to 4.


Hope this helps..

G.Dellaire
McGill Experimental Medicine