Blunt ended ligation not working! - (Nov/07/2007 )
Hi everyone...I was wondering whether anyone has any suggestions for me ??
I've been trying to do some cloning for a while now by blunt-ended ligation. I'm using Beta-gal basic vector (that i have already ligated a 1.6 kb fragment into) and cutting it with XhoI and NheI to remove 800bp of the insert that i put it. I then blunt-end the vector with T4 DNA polymerase supplemented with dNTPS at 16 degrees celcius for 15 minutes). I purify the vector by running it on a gel and cutting out the desired fragment (8.8 kb) and running on a qiagen column. I then go on to phosphatase it with Antarctic phosphatase for 30 minutes at 37 degrees celcius and inactivate the enzyme at 65 degrees celcius for 5 minutes
For the insert, I have already made a clone that, if cut by AhdI and SalI gives me a 7kb fragment that i can purify by running the digest on gel. Before this, I blunt end in the same way that I did with the vector (T4 DNA polymerase).
Finally I have set up ligations (1:1, 1:4 (vector:insert) and control with no insert). The plates looked encouraging the first time round: 2 colonies on control plate, 15-20 on 1:1 plate and around 40 on 1:4 plate. when i picked colonies from both ligation plates, some wierd ligations had happened in which the digestion pattern didn't match any possible ligation!!
The second and third times, I did exactly the same thing and had no colonies on any plates!! I would greatly appreciate any suggestions!!
My best advise is to revise the ligation strategy. Blunt end ligation is not for the faint hearted.
Is it not possible to use alternative restriction sites and conduct a cohesive end ligation?
Could PCR be conducted on your insert, adding useful unique restriction site to the primer's 5' end.
I suppose...The thing is, I didnt want to do a PCR for such a long insert (7kb) due to higher likeliness of mutations being introduced. I've heard that you can introduce mutations even if you use a proof reading polymerase when you're amplifying such a large product!
I also added this to another thread, thought it may be helpful to you too.
In my practice, I never had such problems with blunt end ligation.
I normally use a quick ligation kit from Roche and I keep my ligation reaction for 1 hour at 15 degrees. Never did dephosphorylation. Yet I do find vector self ligations (3 or 5 out of 10) but its ok. and whenever I do dephosphorylation, it surely decreases the ligation efficiency. Furthermore, I almost always keep the vector to insert ration of 1 to 10. and dont make your ligation reaction go beyond 10-20 microliters.
For ligation of large inserts, I usually do an overnight ligation with a normal T4 ligase at 15 degrees.