help: cloning oligos

Ian Graham pdzirg at vme.nott.ac.uk
Mon Jul 3 11:52:19 EST 1995


In article <3t59u9$gva at ari.net>, Soon Paik <paiks at ari.net> wrote:

> Dear Netters,
> 
> In  order to generate a ribozyme expression construct, 
> 1) I have generated two complemetary oligos (49mers) with EcoRI and XhoI 
> overhangs on each end.  So that when hybridized they will be like the 
> following.
> 5'-aattcc----------------------tc-3'
>   3'-gg----------------------agagct-5'

> 6) checked many colonies and cannot find the right inserts.
> 
> What have I done wrong?
> Any suggestions?


Well, Soon, I would consider the most likely option to be that you have a strong secondary structure in your oligos, which is preventing them from annealing to each other, hence not clonable. This is especially likely if you are trying to generate an RNA with a "hammerhead" type structure. The way to get around this _may_ be to put them into ice-water after the 70 deg treatment, then allow them to warm gently to room temp.
Why not mix the two oligos together before kinasing them, so that the heat inactivation of the enzyme and the denaturation of the oligos is combined in a single step?
An alternative suggestion is that any complex secondary structure may need to be removed before the kinase treatment, since the enzyme works well only on free 5' ends. Five mins at 70 deg followed by 3 mins on ice-water should do the trick.
If none of these things work, you may have to clone the sequence in two parts, if that is feasible. That should overcome secondary structure effects, but will obviously take longer.

Hope this helps - good luck!!

Ian Graham PhD
Dept of Genetics, University of Nottingham

ian.graham at vme.nott.ac.uk



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