what competent cells should I use to clone big size DNA - (Jan/17/2006 )

Hi all,

I want to clone a 3.8kb gene into 4.1 kb vector. which kind of competent cells is better for the big size cloning?

Thanks

Hi Stormlina.
Popping a 3.8 Kb insert into a 4.1Kb vector doesn't make it that big of a plasmid to need any special type of bacteria.
Just use the usual competent cells like DH5 alpha or XL-1 blue and everything should be fine.

Hi pBluescript,

Thanks for reply. But I tried many times, it never worked.
I always get strange small band when cutting to confirm the clones. so i am not sure whether competent cells themselves will cut the insert by chance...

If you fear your cells cut the insert, what's your insert like? Is is it DNA with weird structural capacities, or a lot of repeated sequences? In that case I suggest you use SURE or Stbl cells.

Other than that, are you sure about the sequence of your vector and
your insert?

Could you also shed some light on your cloning strategy, maybe it's not the cells but some other step that's going wrong (it might be your cells, but from the information we get here, no one can tell).

Hope this helps.

I agree with pBluescript -- cloning a 3.8 Kb insert into a 4.1 Kb vector is routine, and should not require any special competent cells. As an example, I once cloned a 17 Kb insert into (no kidding!) pBluescript and transformed DH5alpha without issues.

So, I doubt your troubles arise from the size of the clone. The content of the clone is another matter, however...

Provide us with some more information (as vairus suggests), and we'll see if we can throw out some ideas...

Hi guys,

Thanks very much for your suggestion. Actually I asked the question before, and the problem is like this:
I have tried to clone a 3.8kb insert (pcr)into a 4.1 kb and another 5.1 kb vector. For the first vector, I use Sal I to do single cut, for the second one I use XbaI. of course, I deal with the insert the same way.
my steps are:
1. cut both the vector and insert, usually I cut the vector for one hour and then add CIP for another hour, some times I tried longer time for digestion.
2. run gel and do gel extraction using Qiagen.
3. do ligation with T4 ligase, the insert/vector ratio usually is 10.(I usually use 15ng vector). sometime i do the ligation half hour in the room temperature, some time 14 degree overnight.
I use the cut vector only as control.
4. Transformation into DH5a homemade competent cells
each time, I can get a lot of clones compared to the control. the problem is when I do mini-prep and cut the plasmids to check, they are not correct! I tried different enzyme to check...and I usually pick up 10 clones from each plate and one from control plate.

Maybe this is not the problem but just in case it is...
Did you check if the plasmid DNA you are using was prepared from an E.coli strain that has the Dam gene? If these gene is not deleted from the bacteria genome, you will have a problem because the plasmid will be methylated and the XbaI restriction endonuclease enzyme is sensitive to Dam methylation.
But you can verify this experimentally by running your vector single digested with xbaI on a gel.

There should be no problem in cutting a PCR product, since it is not methylated.

Another possibiulity is the UV light:
Before cuting the insert band from gel, you shouldn´t expose it to Uv light to take a picture to it.
Place the gel on top of a glas plate instead of placing it directly on top of the UV screen.
Sometimes the Uv light is too strong, damaging the DNa and afecting the subsequent cloning.

take a picture of gel without the glas only after you cut the band. There is no band anymore, but the picture will be just a reference to confirm you have cut the right band.

Good luck

*sigh*

You are using XbaI.

IMHO, with no proof to back it up, only continual personal and bioforum observation, Xba is the problem.

I don't know how or why.

Xba and Xho... evil.

Sorry I can't be more specific.

Also, do you have any way to guarantee your PCR-product is cut? How many extra bases have you introduced into your primers next to the restriction sites? (Or did you first clone into topo?).
Btw: are you also gel purifying your PCR-product? If your PCR gives one specific band, I don't see why you would do that (I know you can easily ligate UV-lighted insert and vector, but if not necessary don't do it, use column purification if possible).