Ligation issue: Does vector size matter? - (Jun/19/2009 )

I'm facing the problem with a ligation between an 11.5Kb vector and an 1.5Kb insert. Normally, with this size of insert, ligation worked well with me, but in this case, it doesn't. I used sticky end ligation with NotI on the two ends. I treated Antarctic Phosphatase for my vector after digestion, purified both vector and insert by gel extraction and use the vector: insert molar ratios of 1:3 and 1:5. I also performed control experiment with the ligation of only the vector. On both 2 plates, few colonies appeared with the same colony numbers.
The vector I use have the pBR322 origin and the host is XL1-Blue.
I heard that over 10Kb, the ligation with pBR322-based vector becomes problematic, is it true?
Every suggestions are highly appreciated, thank you all in advance!!!

It is the second ligation reaction that makes it difficult. Once one end of your insert is ligated to one end of your vector, it's a probability curve. What are the chances of a 13 kb linear DNA molecule finding its own other end versus finding the end of another 13 kb linear molecule or another 11.5 kb linear molecule or another 1.5 kb linear molecule or the end of some other linear concatamer?

You said "a few colonies appeared". Did you check them to see if any had an insert?

Is there a way you can do the ligation using two different enzymes?

We routinely do insert + vector and three-way ligations with a 10.5 kb vector with a pBR322 ori, so it's not impossible...

thank you for your reply. I did check every colonies I've got, some are the self-ligated ones, some ligated to a concameter, not my insert T_T. Using NotI is my only choice. Base on what you say, I guess using a higher incubation temperature may increase the chance for the "second reaction" to happen, rite? Normally I use 16-18 Celsius degree, maybe room temperature can help?

Quasimondo on Jun 20 2009, 12:01 AM said:

to me, higher temp didn't make a great difference and sometimes even worse. I routinely do ligation in 4C for at least 24h, the ligation efficiency is much higher. But I don't know if this will work in your case. I haven't handled such big vector.

Nrelo on Jun 22 2009, 12:14 AM said:

I have the answer for my problem, it is the antarctic phosphatase, I overused this enzyme, so it damaged the ends of my vector. I reduced the amount of enzyme treated and positive colonies were detected, even at not high percentage (3/20) with one has the desired orientation of the insert. I perform the ligation at both 16 and 25 degree, but the RT sucked, no correct colony was detected. Thank you all for the replies.

I routinely dephosphorylate my vector using 1/10 the recommended amount, and for only about five minutes, then straight on a gel for purification. We use calf intestine alkaline phosphatase.

HomeBrew on Jun 24 2009, 11:49 AM said:

Thank you, now I know my problem. Usually we use CIP but recently we didn't buy the new one, so I have to use the AP instead. Next time I will use the procedure as you mentioned, maybe my routine one is not good, it reduces significantly the ligation efficiency.

Quasimodo,

Do an ethanol precipitation..

Add ur " gel extracted DNA" and the dephosphorylated DNA. then do ethanol precipitation . resuspend in small volume and run ligation.

i m pretty sure that will give u something cool

gel extracted DNA is nasty~

might survive PCR but mostly not ligation

yes, as the vector becomes larger, the ligation does become more difficult. As mentioned by the Homwbrew, it is the probability of the ends of the ligated molecule meeting. When the ligated molecule is short, the ends of the molecule are effective close together. The two ends of a short linear DNA molecule can not wander far. Contrast this to a long linear DNA molecule.

In general, some difficult is first encountered once the ligated DNA molecule exceeds 10kb.

If you do gel extraction of your vector, try to avoid or minimize exposure to UV light. It really does reduce the probability of obtaining your desired plasmid.

You could also reduce the DNA concentration. Lower concentration increases the probability of intra-molecular ligation, as opposed to inter-molecular ligation.