Cloning big inserts - (Dec/06/2007 )

Hi all!
I'm trying to clone a 4,2kb insert into pET system, but I'm having a hard time! I get transformants, but wrong size or no insert at all. The negative control is fine, I mean, the plasmid is not re-ligating.

Any tips are welcome!!!

Thanks to all,

Isabela.

Hi Isabela,

Are you certain that your ligation is working such that your adding full-length construct into competent cells? Do you know if your gene is toxic to E. coli? If you can, try transforming several different E. coli strains. I've found that what didn't clone in one strain does clone in another.

Regards!

i think it would be helpful if you could tell us more about what the nature of your insert DNA (is the DNA made of tandem repeats, is the vector expressing the insert? the type of protein your insert express etc)

As describe your ligation strategy. What enzymes you are using, how long are you cutting for, and how much DNA are you cutting.

Describe exactly in painful detain what you have done to your DNA, what control have you conducted, what gels you have run. Did you check to make sure all you insert was the same size? How about the duration of exposure to UV? Long UV exposure when excising your gel bands is very very bad. Did you check your insert and vector once it was cut on a gel? What protocol you have used, how long? Include the digestion formula you used and the formula's of the ligation reaction, dephosphrylation etc....

It is the details that influences whether a ligation will work or not. A lot of things can go wrong so more info will help find the sourse of the problem. Else all we have will be wild guess.

Hi there!

I believe the ligation is working, because I'm doing several other different ligations at the same time and they're perfect. I the gene is toxic, no clue, but I already suspected that as well. I'll try to transform some other strains! Thanks for the tip.

Regards,

Isabela.

Hi there!

My DNA is a full gene for expression (it is a dehydrogenase)

I'm cutting with NcoI/Mung bean/BamHI, separately of course. The restriction incubation is overnight for each (except mung bean).

As I mention in the other reply, apart to the digestion, everything else is the same for some other ligations that I made, that worked perfectly (gene cleans, checking purification, ligation conditions, competent cells). To gel-purify I'm using Roche kit, and checking afterwards. Ligation is run at 16oC o/n.

I don't really dose the DNA I digest, for I use some eye-o-meter...

Thanks for any tips in advance!

Regards,

Isabela.