Multiple fragment ligation - (Jun/20/2007 )

I think that when ligating, we always have 2 fragments - a vector and insert. I was wondering, has anyone ligated 3 pieces of DNA together in the same ligation reaction? All fragments have the correct sticky end restriction sites, so the 3 pieces should only be able to fit together in 1 way. In theory it sounds ok (to me), I just wondered if anyone has done this in practice.

If 3 does work, would 4, 5...6? I assume that the greater number of fragments would reduce the efficiency of ligation and getting correct clones after transformation?

QUOTE (miRNA man @ Jun 20 2007, 04:48 PM)

I think that when ligating, we always have 2 fragments - a vector and insert. I was wondering, has anyone ligated 3 pieces of DNA together in the same ligation reaction? All fragments have the correct sticky end restriction sites, so the 3 pieces should only be able to fit together in 1 way. In theory it sounds ok (to me), I just wondered if anyone has done this in practice.

If 3 does work, would 4, 5...6? I assume that the greater number of fragments would reduce the efficiency of ligation and getting correct clones after transformation?

Yupper, multi-ligation certainly works! There are a number of multi-ligaters here.

The greatest number I have done is 5 ways. Efficiency is reduced with multiway ligations, but colony PCR with multichannel pipettes can easily compensate.

I guess 7 or 8 ways, might be the limit using colony PCR.

Lets have an example of a 4 way ligation. There is

vector V
fragment A
fragment B
fragment C

Firstly all fragments, vector and inserts must have incompetible ends. Thus the fragment can only ligate together in only one possible arrangment. If you do have a fragment that can go in two orientations, the probability of finding the right plasmid falls in half.

Importantly, as this is multiway ligation, you are very likely able to build any construct in a single step from an empty colour testable vector. Ie pBluescript, pTRC99A, litmus28 etc. Having blue/white colour testing is a huge help.

The mol ratio I use for the ligation for V:A:B:C is 1:1:1:1. I have tried doubling the insert ratios, but have not seen any visible difference. The amount of vector I typically use is 400-500ng. It is important to keep the DNA concentration on the higher end. The ligation is better that way.

Ligation conditions I use is overnight incubation at 16 Celsius in a 20ul volume. (It is easier to pipette larger volume; I don't have a p1 or a p2).

The ligase I use, is a standard T4 ligase and normal ligase buffer. Do not use quick ligase, or any ligase buffer that promisese fast ligation. The effect of quick ligase buffer on multiway ligation is horrendous.

Another point to make is the plasmid size. Multiway ligation on smaller plasmids is far easier then conducting a multiway on large plasmids. It is my belief that the difficult of multiway ligation start to increase when the final plasmid size exceeds 10kb.

The efficiency of obtaining the desire clone, depends on the March number of the multiway, your skills at the blackarts of ligation and dumb luck. It is hard to give efficiency values. I have had some superbly prepared vectors, then even when conducting a 3 way ligation gave me an efficiency of better then 1/2! Other times, I have made really bad vectors, which left me seaching 100 colonies (also a 3 way) to find a single positive clone. And the dumb luck bit comes from the random nature of probability. You may search the first 50 colonies and get nothing. But the next 50 yeilds 5 postive colonies.
Probability is thus 1/10, but seraching 10 colonies will never give you a single positive clone.

A multichannel pipettes, 96 well plates, colony PCR and PCR plates are an instant must for multiway ligation. It is the easiest, fastest way of screening many colonies.

But to give at least a vague idea, I listed the my personal efficiency of recovering a colony with the correct plasmid.

2 ways: 9/10
3 ways: 1/4
4 ways: 1/25
5 ways: 1/72

The average number of colonies I screen is 72. For 5 way ligations I would screen 84 colonies, easily going as high as 168.

which primer do you use for conoly pcr?? is it gene specific primer or any primer that amplify insert of pT7 of pUC??

I usually make diagnostic primers to do the colony PCR. Each PCR fragment type usually has its own diagnostic primer. Small short primers are relatively cheap.

Thank you - that is very helpful! What are the false positives, empty vector or incorrectly assembled constructs?

The false colonies, tend to contain empty vectors that have recircularised in some oddway.

Colonies that give a positive signal on PCR tend to have their inserts assembled in the correct manner. However there is a probability of incorrect assembly. It becomes noticeable at 4 ways and a problem at 5 ways. So for 4 and 5 ways, I usually screen my clonies twice by PCR to get the right clone.

But in anycase, I always midiprep 2 to 3 colonies and then test the plasmid by restriction digest to make doubly sure