blunt-sticky ligation/transformation problem - (May/13/2005 )
I'm trying to clone a Hpa (blunt) AvrII (sticky) fragment into an Hpa/AvrII digested vector. The insert is only 2kb while the vector is ~11kb. I know that the vector and insert both cut fine (the sites on the vector were 900bp apart so I saw an appropriate size band drop down after digestion).
I used a 3:1 molar ratio of insert:vector, also did 1:1. For all ligations, I used 60 fmol of vector DNA ends (roughly 200ng). The ligation was in 20ul using 1ul NEB T4 ligase, which has worked well for me on previous ligations (sticky-sticky). The ligation went at 16 degrees O/N.
I transformed using One Shot TOP10 chemically competent cells:
Plate1: Uncut vector = lawn of colonies
Plate2: Cut vector, no insert = no colonies
Plate3: Vector+insert ligation 1:1 ratio = no colonies
Plate4: Vector+insert ligation 3:1 ratio = no colonies
In all transformations, I followed the protocol exactly (50ul of cells used) and plated 50ul (of 300ul total) on each plate.
Plate1 tells me that my cells are competent, so I know that's not the issue.
I am currently running the ligation on an agarose gel to verify that the ligation worked.
Any suggestions? I've heard that chemically competent cells do not transform larger plasmids as efficiently as electorcompetent, is there any truth to this?
Also, should I kinase the insert?
Finally, is there something wrong with ligation conditions? I've thought about getting some of NEBs concentrated T4... is that needed?
Any suggestions/help would be appreciated!
Hi Paladin,
Can't help much b/c I'm experincing the similar problems. But in my past experience with ligations, doing a phosphorylation on even RE-cut insert helped. Although theoretically the digested ends should have P already.
My ligations also showed no colonies, although transformation +ve showed tons. But my insert was PCRed and digested. Guess I have to try PCR again using promers with more extra bps on each side, so it will be digested efficiently.
How can you verify if the ligation was OK by running it on a gel?
Also, what's the advantage of NEB's high conc. T4 ligase?
Can't help much b/c I'm experincing the similar problems. But in my past experience with ligations, doing a phosphorylation on even RE-cut insert helped. Although theoretically the digested ends should have P already.
My ligations also showed no colonies, although transformation +ve showed tons. But my insert was PCRed and digested. Guess I have to try PCR again using promers with more extra bps on each side, so it will be digested efficiently.
How can you verify if the ligation was OK by running it on a gel?
Also, what's the advantage of NEB's high conc. T4 ligase?
For cloning PCR products, I am now converted to using TA cloning. It's highly efficient and once you've generated the clone, you don't have to ever repeat the PCR again. Also, since the product is now in a vector, you avoid the uncertainty of whether or not the PCR product was digested or not.
Running a ligation on the gel can show you if the ligation itself worked. There are several ways to do it... You can cut the ligation reaction with an enzyme that should linearize the final product (but not one of your cloning enzymes). Examine the gel for the appropriate size fragment. Another method is to run an aliquiot of the raw ligation, if the ligation worked, you should see the insert band disappear (run against ligation without ligase). Alternatively, you could examine the gel for supercoiled DNA. Because there are several ligation posibilities (vector-vector, vector+multiple insert, insert-insert etc), the supercoiled DNA will likely look like a smear.
High concentration ligase. I've read that blunt ligations require a higher concentration of ligase, but I don't have much experience with it. I've also heard that blunt ligations fair better with supplimented ATP. I was just throwing ideas out there, hoping that someone with more experience could shed some light on it!
Thanks a lot, paladin. Will try a cloning vector for PCR.
Re: digesting ligations, is the buffer for ligation compatible with the REs?
Re: digesting ligations, is the buffer for ligation compatible with the REs?
RE buffer not applicable here, the fragments were gel purified. Sorry I forgot to mention that!
I ran the gel and it appears that one end of the ligation worked and not the other. I know this because I ran raw ligation and got two distinct linear bands, one was vector only, one was vector+insert and there was no band corresponding to insert alone. This tells me that one end ligated, but the thing didn't circularize
I'm guessing the blunt end is the culprit! Not sure what I'm gonna do with the ligation... I'm thinking about spiking it with some fresh T4 ligase, add some ATP (if I can get my hands on some) and let the puppy ride over the weekend at 16 degrees...
Hi!
You can do two things:
one is try to ligate your insrt(blunt and sticky) with desphosphorilated vector or second, why don't you try to make blunt both ends. Make blunt your insert and also your vector with the Klenow enzime (USB) and later desphsophorilate your vector. For blunt ligation you have to make a bigger ratio, almost 1:6 for vector insert. The problem is that you have to begin with a big amount of insert because in the purification step you always loose some insert. Is very important to begin witth a big amount of insert 'cause your vector(11kb) is almost 5 times bigger than your insert (2kb) and the ligation reaction have to be 1:6!!!!!Remember!
When you make a blunt ligation sometimes you need to add ATP (take care when you thaw ATP, if you freeze and thaw too many times it can't be degradated, make aliquots), but it depends on the manufacture's protocol. I normally use T$DNA ligase from USB and it works very well.
At last, when you transform your ligation you could do making heat shock, but sometimes when you have big vector (around 12-15kb) the efficient is reduced and you obtain few colonies, but is not impossible 
You can try by electroporation but I always do by heat schock and don't have problems. 
Good luck!!!!!!!
Baobab
You can do two things:
one is try to ligate your insrt(blunt and sticky) with desphosphorilated vector or second, why don't you try to make blunt both ends. Make blunt your insert and also your vector with the Klenow enzime (USB) and later desphsophorilate your vector. For blunt ligation you have to make a bigger ratio, almost 1:6 for vector insert. The problem is that you have to begin with a big amount of insert because in the purification step you always loose some insert. Is very important to begin witth a big amount of insert 'cause your vector(11kb) is almost 5 times bigger than your insert (2kb) and the ligation reaction have to be 1:6!!!!!Remember!
When you make a blunt ligation sometimes you need to add ATP (take care when you thaw ATP, if you freeze and thaw too many times it can't be degradated, make aliquots), but it depends on the manufacture's protocol. I normally use T$DNA ligase from USB and it works very well.
At last, when you transform your ligation you could do making heat shock, but sometimes when you have big vector (around 12-15kb) the efficient is reduced and you obtain few colonies, but is not impossible
You can try by electroporation but I always do by heat schock and don't have problems.
Good luck!!!!!!!
Baobab
Thanks for your suggestions! Unfortunately, I can't do blunt-blunt because I'm cloning part of an ORF and I need to keep things in frame and I can't change amino acid sequence! I will definitely try a 1:6 ratio though and add ATP to the reaction!