blunt ligation of large fragment - (Oct/09/2008 )
Hi 
,
could you please help me with ligation of a large product?
I try to ligate 2,7kb large PCR product to 3,1kb large pJET vector (blunt ligation) and I have plenty of colonies but the vector is empty. 
Later I would need to subclone the gene to another vector but that will be based on sticky ends.
The PCR product was amplified with Phusion HotStart Polymerase and cutted off a gel. The vector is linearised and (that is also strange) it shouldn´t be empty as it carries some gene that is lethal for bacteria. In my former ligations I also got "empty" vector but it never was a plaque as I can see it now but only a few colonies among the good ones and the vector carried some mess. This time there is just the 3kb vector.
Are there any tricks how to blunt-ligate fragments of the same lenght?
Would you think that to cut the PCR product straight after amplification with restriction enzymes and ligate it with the final 4,7kb expression vector woul work better?
Thank you for any answer
I've been trying to do the same thing, although in a larger vector. And my product is does not come from a PCR, but from another vector. The insert has PmeI sites at both ends.
Any hints would be welcome!
Concerning blunt end ligation my advice normally is
"Avoid blunt end ligation if possible. They are more difficult to do compared to sticky end ligation. Please revise ligation strategy."
However you are using pJET vector (Fermentas), which carries a lethal gene that should give positive selection for insertion.
Hmmm... well you could try using ligation buffer with a final concentration of 10% PEG6000 (ie a quick ligase buffer). The PEG does improve blunt end ligation efficiency.
You could also conduct your ligation at 4 Celsius, overnight. Lowering the ligation temperature sometimes helps.
Increasing the insert to vector ratio from 1:1 to 1:3 can help. remember it is important not to use too much insert and it will cause ligation problem (ie the vector ligates to 2 insert molecules and as a result can no longer religate.
Test more colonies. I find it a little strange that the eco47IR gene isn't working so well. Please check that your PCR insert is okay, pure and not overly exposed to UV (when you are excising the band from a gel matrix)
"Avoid blunt end ligation if possible. They are more difficult to do compared to sticky end ligation. Please revise ligation strategy."
However you are using pJET vector (Fermentas), which carries a lethal gene that should give positive selection for insertion.
Hmmm... well you could try using ligation buffer with a final concentration of 10% PEG6000 (ie a quick ligase buffer). The PEG does improve blunt end ligation efficiency.
You could also conduct your ligation at 4 Celsius, overnight. Lowering the ligation temperature sometimes helps.
Increasing the insert to vector ratio from 1:1 to 1:3 can help. remember it is important not to use too much insert and it will cause ligation problem (ie the vector ligates to 2 insert molecules and as a result can no longer religate.
Test more colonies. I find it a little strange that the eco47IR gene isn't working so well. Please check that your PCR insert is okay, pure and not overly exposed to UV (when you are excising the band from a gel matrix)
Agree with persene, try revise if possible. Blunt end is at least 100-1000 less efficient than cohesive end.
In addition my Genetic Engineering prof used to tell me that increasing ( overkill) the reaction with more more T4 ligase will do the trick too.
WISH U LUCK. but if u have a method of visual screening or selection that would really make the process go way faster in searching for colony with insert! Make sure u are using ligase from T4 and not e.coli sourceby the way!!
The insert ends are phosphorylated right? If not, that must be done. As said previously, blunt end ligations are difficult, as are large fragments, so you have a difficult ligation to begin with there and a sticky end strategy would be more helpful. Blunt end ligations, like all ligations, are encouraged by increasing molecular crowding (i.e. bringing everything closer together so they can react better with one another). There are several things you can do to increase molecular crowding, some of which have already been mentioned:
-more units of ligase - more enzyme molecules reacting with the DNA ends
-PEG-8000 - takes up space in the reaction and squishes everything else together
-more vector and insert - unlike what Pernese says, in your case i would add more insert and maybe less vector. Ligation appears to be happening (empty vectors) but it's just your insert is not ligating into the vector. So i would add as much insert as possible to cram it in there. In fact, in your case i would add the various components of the reaction and then make up the rest of the reaction with insert solution (i.e. don't bother adding any water). Given that your insert is large, the molar ratio of insert:vector will also tend to be lower due to the size of the insert, so the more insert the better.
-smaller total reaction volume - keeps things concentrated
Ligations can occur with any incubation time and temperature but for difficult large blunt-end ligations, longer incubation times and lower ligation temperatures tend to be more helpful.
Thank you very much all your advices!!! 
I choosed the pJet vector two years ago, when I came to a new lab with a narrow budget. Till now I didn´t have serious problems with it. Hope to overcome this one too. Also good luck to Madrius!
Hehe, thanks!