Please help me! my ligation is fail - (Dec/07/2005 )

Hi
I have some problem with my cloning. At first, I cut my vector (pcDNA3.1, 5.4kb) with EcoRI and NotI. After that, I run gel to check my digested vector and it seem to be good ( I got one band about 5.4 kb, b/c it was cleaved about 25-30 base out) and then purified with spin column (nucleospin).
Also with the insert, I cut the insert from my plasmid with EcoRI and Not I, run gel and cut my target band (5.1kb) from gel.

After that, I performed ligation with T4 ligase, incubate overnight at 4C. I transformed my ligated into DH5alpha completent cell and plate on Kanamycin plate. I got a lot of colonies and i selected them, did miniprep and digested again with Not I (check for the right clone). Normally I should recieve 1 band (about 10.5 kb). BUT I got just only 1 band about 5 kb !!!!!!!. My ligation was fail and I dont know what should be the problem?

Please give me some suggestion, b/c I stuck in this problem more than 3 weeks.

Thanks in advance

Kate

-kate_ja-

You can phosphatase your vector to reduce background. It seems that youÂ´re getting religated plasmid. And adjust the vector:insert molar ratio.

By the way, IÂ´ve been stuck in a cloning for seven months now, so donÂ´t complain for three weeks!!

-ger225-

Hi Kate_ya,

Why do you incubate your ligation mixture at 4 degC overnight? I thought T4 ligase requires a 16 degC incubation overnight????

-cska_fan-

I think you should recheck your vector:insert ratio and try and make some adjustments. Good luck!

-nky-

Dear Kate

You should de-phosphorilated your vector after digestion to avoid re-ligation. I always use Bacterial Alkaline Phosphatase (BAP) for de-phosporilation treatment. After the treatment at 37C about 20 min, inactivate the enzyme using incubation at 65C for 20 min. Then, phenol-chloroform and etOH precipitation were recomended to get the clean digested vector. Finally, measure the concentration of the vector and insert before ligation.

Good luck

-Ali-

QUOTE (kate_ja @ Dec 7 2005, 06:14 PM)

As other people have said, you have to check more carefully your insert:vector ratio, it's a tricky thing and it will take time to get a better knowledge of amounts you have on the gel. Second, I wouldn't use Not I since that is the enzyme you've used to digest with and it's better to use one unique to the fragment but not to the vector, meaning the one that cuts just once in your fragment and doesn't cut the vector. Then you will be sure that your fragment is really there. Or just take some primers if you have some created that are inside your fragment and run the PCR and look for the expected size fragment. But digestion is always a better option since it's more of a proof that you have a fragment on your plasmid. Another thing, it IS really strange that you are ligating O/N at 4 degrees, T4 ligase should be left on room temp. over night or even just 2h are enough. Be sure also you are using the right buffer, meaning 5X vs. 10x. Good luck!

-smoochiepie79-

When you cut your vector, how can you be sure both your enzymes have worked? Just linear vector and not double cut vector can not be distinguished on gel, so cut your vector with either one of the enzymes and check if it is really linearised. For your target gene, how big is your plasmid you're cutting it from? Have you run uncut vector on your gel (just to see where the supercoiled plasmid is going, you never know if sume uncut plasmid would migrate at the same rate a your gene of interest). Apart from this, I suggest what other people have said here.

-vairus-