Ligating large fragment into vector
dellaire at ODYSSEE.NET
Thu Jun 27 07:40:45 EST 1996
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
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..
McGill Experimental Medicine
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