Cloning failure

[alexis]

Hi everybody!

I'm trying to substitute a 1 Kb promoter, from a bidirectional expression cassette, with another of 644 bp, from another vector.

I've tried many times and I've done all the controls (efficiency of ligase, competent cells, restriction enzimes, ecc..): everything work, but every time I've got no colonies!!

what could be the problem??

I'm at the first experiences with cloning and I'm desperate

thanks to all!

alexis

 

[CharonY]

Being desperate is a normal state of mind when it comes to cloning. So you have done all the controls the only thing would be to check whether everything for your construct is correct (i.e. compatible restriction sites), I am assuming you are excising the promotor from one vector and cloning it into another?

You could play around with concentration ratios of insert to vector.

 

However, there is also a question of the promotor strength and what is being controlled. In some cases overexpression of certain genes may prove to be fatal. In that case it is beneficial to use a promotor that you can repress, although this may defy the purpose of what you are trying to do.

[alexis]

Yes, I used the same enzimes to excise the original promotor from the bidirectional cassette, and to amplify the other promotor from its vector.

I've used the same procedure to clone a 4.7 Kb insert into a 3.5 Kb vector and everything was OK, so this time I can't understand the problem using a smaller insert!

 

Before starting the subsitution of the original promotor, I've checked the activity of the new promoter, and it was a not so strong promoter. I can't think that the new promoter is fatal, because the original promoter is a very strong one, it has been tested and there were no consecuences for the cells.

[CharonY]

So you amplified the promotor? Did you make sure that the amplificates had the correct overhangs (e.g. if you use restriction enzyme have you made sure that the restriction site is not right at the end of the product, for example?).

 

I assume that your ligation mix contains the cut vector, product with appropriate ends and the original (excised) promotor? Have you done anything to prevent re-ligation (and if not, are there any clones with the original vector?).

[alexis]

I've chosen enzimes that produce sticky ends, and I used the same to cut the original vector and to amplify the promoter I want to clone, I don't know if it's clear the procedure I've followed.

 

I don't know if my vector can religate, but the restriction site are different, so it's possible to religate even with different restriction sites?

 

the problem is that I've got no colonies every times and I cannot verify the presence of the vector religated alone. because I've excised from this vector the original promotor, so if this vector is able to religate, without the promotot it cannot produce the Amp resistance, so I'vegot no colonies and I can't understand how to go on