Help! Suggestions for ligation ratios - (Jan/14/2008 )

This website rocks!

Now to my question....I've been trying to perform a ligation with a 16 kb vector and 3 kb insert (they have sticky ends....and yes, I'm quite aware how big these sizes are). So far none of my ligations have worked. I've used TSAP to dephosphorylate one end of my vector to prevent religation. Afterwards I use Epicentre's ligation kit to perform my ligation. I have tried using a insert : vector ratio of 3:1, 4:1, 5:1, 6:1 and am not getting any colonies from my transformation. I use about 200 ng of vector for each ligation. My transformation controls are good which means I don't have to worry about my cells. Does anyone have any suggestions for the ligation ratios. Please help. Thank you!

Pedantic point: the use of a phosphorylase should affect both ends of the plasmid, not just one.

The fact that you don't get any colonies could be caused by leaky, or unintended, induction, especially if the insert codes for a toxic product. What expression system are you using?

What about your other controls? Is your ligase working properly? Test this by quickly ligating some DNA size markers (say, lambda/HindIII, minus the loading buffer, of course!) and running the product on a gel. If you get a high MW smear, the ligase is OK. Next, is the RE step cutting both ends of the insert to a high level? Repeat the quick ligation trial on a bit of the digested insert. The same rules apply: anything other than a smear at high MW means there's a problem somewhere.

The 3kb insert does not encode a toxic product. It is part of a viral genome that encodes structural proteins. The original 3kb fragment of the 19 kb plasmid had a duplication in it. I am replacing that fragment with an identical fragment that does not have the duplication.

The lab actually ordered a new Epicentre ligation kit. I will use that kit once I have my vectors and inserts prepared. I'm also having trouble extracting my DNA from the agarose gel. I am removing the 3 kb fragment from the 19kb plasmid by digesting the plasmid with BstE II. I'm running the digest on a 0.7% agarose gel to isolate my 16kb vector. I'm using Qiaex II to extract my DNA. I am digesting about 400 ng of DNA per reaction, but after the extraction I have about 10 ng/ul of DNA left. Does anyone have any suggestions on how I can increase the concentration of DNA during the extraction procedure.

Any suggestions on the ligation ratios?

I find it hard to believe that you can tell whether you have 10 ng of DNA or not, without running all of it on a gel. Do you perhaps mean 10 ng/ul? This is plenty of DNA to ligate -- use 5 ng of your vector and mix them. How are you transforming? The large constructs like this can be difficult to transform efficiently, even with highly competent cells. But my first bet is that you are damaging the insert with UV during gel extraction. Use long wave (365 nm) or blue light for transillumination and expose for a minimum amount of time.

How long do you digest the plasmids with the enzymes. Digestion for extended time periods can damage the ends and you will get no colonies.

Try to have 20-30 ng in ligation and have some thing like 1:3 -1:5 ratio and give it a go. And how much of the ligation do you use for transformation?

200 ng of a 16 kb vector = 19.23 fmoles of vector or 1.16 e 10 molecules.
10 ng of a 3 kb insert = 5.128 fmoles or 3.09 e 9 molecules.

All of the 1:3, 3:1, 10:1 ratios of insert:vector are shortcuts to convert nanograms of typical inserts and vectors to an equal number of ends.
What you really want are equal fmoles (or number of molecules) in the ligation. If you try to ligate 30 ng of a 3 kb insert with 10 ng of a 16 kb vector, your ratio is 9.3 e 9 molecules of insert:5.8 e 8 molecules of vector or 16:1 in number of ends.

53 ng of a 16 kb vector = 3.07 e 9 molecules, so combine 53 ng of your vector with 10 ng of your insert (or better yet: 5.3 ng of vector with 1 ng of insert) for a roughly 1:1 ratio of number of ends.

When extracting DNA that is greater than 1 kb from agarose, do not delay after stopping the electrophoresis. Yields are higher if you extract before compounds in the agarose have a chance to bind to the DNA.

Just reiterate the point made by tfitzwater,
Ratios of insert to vector is not a key point to a successful ligation. It is the mole ratio, the ratio between number of molecules that we are using, not the mass ratio. As long as the ratio of vector to insert molecules is about one to one, your ligation should work.

The key problems to ligation as stated by phage434, scolix and swanny is the quality of your DNA, the state of your ligase enzyme and only when working with large plasmids, the competency of your competent cells.

T4 ligase is notorious for going bad. So one has to check on it ever so often.

As for DNA quality, big fragments easily fragmented if the DNA preparation is poor, over exposure to UV is also particularly severe as the number of molecules you are working with given a particular mass is reduced. Probabily of UV induced cross linking increases with large DNA fragments. Thus it is best to avoid exposing your DNA to UV. What I do is run my DNA on a gel, then cut a frame, which contains the DNA ladder and the ends of the DNA band which I have size fractionated. Exposed that frame to UV, and mark when the ends of my desired DNA band is. Put the frame back to gather. With the guide of the markings on the frame, cut out my desired DNA band.

As already stated, when working with fragments this big, you will need so very competent cells. Such as Oneshot genehog from stratgene. Really large fragments do not ligate well and don't transform well either.

I just want to say thank you to everyone for the helpful suggestions. I've only been working on this project for about four months so things are still pretty new to me. I appreciate all this help