Big fragment ligation - (Feb/06/2008 )

Hey guys...

I ve been trying to clone a 5.5 kb fragment into a 4.8 kb vector without any success. Apart from the molar ratios and dephosphating techniques (which i don t mind hearing some opinions about), do you think it s possible to ligate a fragment of this size to this vector? (pGL3 , Luciferase vector). Also, do you think it s possible for the competent cells to take up such a large plasmid? Should i use electrocompetent cells? Do you think subsequent transfection of mammalian cells with a construct of this size is feasible?

Thanks for any advice you may have.
Vicky

yes, you certainly can ligate that together. Don't despair. It can be done.

If you tell us exactly what you did (we need all the details you can give), somebody could hopefully point out where the problem is hiding.

This should be straightforward, with few difficulties. You want to use lower DNA concentrations than you think during ligation to optimize recircularization rather than concatamerization. 10 ng of insert and vector are plenty. Ideally, you want to do this with two enzymes and avoid the dephosphorylation step. If you can't for some reason, then dephosphorylate with SAP or antarctic phosphatase and heat kill. Don't overdo this reaction -- follow the instructions. Do a no-insert ligation control to quantify the success of your reaction. Transformation should be straightforward with either chemical or electroporation techniques.

Can be done. Folow the instruction by phage434 and indeed tell us what you've done (what enzymes, did you gel purify, any controls for competence of your bacteria, negative controls etc). To give you some hope: i've done more than 20 5 kb insert and 10 kb vector ligations, where I had the right clones the first time in all cases! (restriction digested and sequenced to control).

And with my 15 kb constructs I can transfect mammalian cells (293T, Hela, U87 and MT-4 are the ones I tested, so there's no reason why others can't be transfected with such plasmids).

Hi again.
Thanks a lot for your answers.
The first time i did a standard ligation, using a non-dephosphated two-enzyme digested vector and a vector/insert ratio 1 to 2. I got too many background colonies in the control plate and those i screened from the other plates had no insert. Then i tried dephosphating the vector for 15 min with SAP, which i subsequently heat-inactivated for 15 min at 65 C. I got no colonies in any plates after transformation. After that, i did another ligation without dephosphating, but with a re-digested and re-gel purified vector just to get rid of any undigested stuff and i used a vector/insert ratio 1 to 5. Strangely i had no colonies in any plates. The things i changed comparing to the first time was the total DNA amount (about 10 ng of vector, first time about 100 ng) and also the fact that i used the whole 20 microliter ligation reaction for transformation, whereas i usually use half of it. I ve done ligations with smaller fragments in the past and they usually worked, although at a low percentage of colonies i have to say (about 20 percent).
This time i completely dephosphated the vector again, but performed a phenol/chloroform extraction after heat inactivation to get rid of SAP possibly inhibiting the ligation/transformation and i 'm using a 1 to 3 ratio and 30 ng of vector in the transformation. I haven t got the results yet. What do you think?
So you guys really think that standard chemically competent top10 cells are ok? I was thinking that that might have been the cause of failure of my transformations in the last two experiments.

Vicky

One of the problems you have is that you are using too much ligation reaction in the transformation. 1-2 ul is the sweet spot, not 10-20 ul. Large amounts are inhibitory. Are these commercial Top10 competent cells, or ones you have made yourself? What transformation efficiency are you getting with the controls?

What enzymes are you using to cut the vector and insert? You should check that the enzymes cut the vector individually.

Can you control for double digested versus single digested vector? If enzyme sites are close to each other you will not notice the difference (and gel purification doesn't help either then, you might only damage DNA by UV-exposure then).

Never go above 10% of total volume of competent cells for transformation(assuming you use 50 µl of commercially available cells: don't go above 5 µl, 1-2 is better).