You have 50% chance to have your insert in the correct orientation.  So why are you complicating the task just do your cloning and screen for the right orientation using PCR and pair of Primers one on the vector and one in the right direction of your insert. All the positive clones will give a band with the expected size

Wishing the best

Thank you for your response.

Major problem with single enzyme digest is getting most of the colonies of vector self-ligation. Only few colonies will have your insert and again only 50% chance of getting in right orientation. If you CIP the vector, you can solve the self ligation problem.

I'd be tempted to sequence my clones to make sure the orientation is correct and also to ensure there is no unintended mutations in your clone.

When using a single r.digest site for subcloning you cannot predict or encourage the direction of ligation. But what you can do to prevent false positives (religation of your plamids after cutting with the single enzyme) is to dephosphorylate the plasmid and phosphorylate your insert. two options you either phosphorylate the PCR product or phosphorylate your primers before you carry out PCR- this approach is more efficient. Your can also purchase phosphorylated primers but they are a bit more expensive.

Thank you everyone for your inputs! Yes, I always dephosphorylate when using enzymes with compatible ends or blunt cutters. I think I need to simply screen more colonies. I did a restriction digest on DNA from 10 colonies yesterday and they all show the cloning worked, albeit in the incorrect orientation.

I'm very surprised that after testing 10 clones all of them were in the incorrect orientation!! As stated by the colleagues above you should expect a 50% of colonies in the correct orientation. This is really random cloning.

This make me think that perhaps the insert is toxic  for the bacterial cells (or produces a toxic product).  Do you know which is the product encoded by your insert?

In these cases I change of bacterial strain and leave the colonies grow for longer in the plates, just in case the insert only delays the growth and do not kill the cells.

Hope this helps

You can try to digest the cloned vector with an enzyme that has one site inside the ORF (better in the extremes of the ORF) and another that has the recognition site not in the orf and then in silico you can predict the size of the digested bands (note that the different orientations will give different sizes of bands) so then you pick the one with the right orientation.

If you're only getting the 'wrong' orientation - presumably meaning that the promoter transcribes the antisense strand - then it suggests that your product is toxic when overexpressed. You will need to keep the gene 'turned off' until it is time to induce it. 

If for example, you are cloning in a plasmid of the pUC family with the gene under control of the lac operon, keep the lac operon turned off by adding glucose and leaving out IPTG. The drawback with this is that you lose the blue-white screening, but if you're using dephosphorylated vector this shouldn't be too much of a problem. 

Another trick you might like to try is to deliberately pick small colonies. This will work if your product is mildly toxic and reduces growth rate without actually killing the cells. 

Andrew Jenkins i don't understand " The drawback with this is that you lose the blue-white screening, but if you're using dephosphorylated vector this shouldn't be too much of a problem. " can you explain more clearly?

In a pUC vector, the insert interrupts the lacZ gene, resulting in loss of expression. In the presence of IPTG (the inducer) and XGAL (a chromogenic substrate), colonies without an insert are blue, while those that have an insert are white.

I was suggesting that you leave out the inducer, IPTG, and add glucose, which further reduces expression from the lac promoter via catabolite repression. This would prevent a potentially toxic product being produced from fusions with the lac operon, but as the lac operon is switched off, colonies without an insert will no longer express lacZ and you will not be able to use the blue colour produced by cleavage of XGAL to distinguish between colonies with and without insert (recombinants and non-recombinants).

Non-recombinants arise from recircularisation of the vector; this is prevented by dephosphorylation, as two dephophorylated ends cannot be religated. In theory, all your colonies should be recombinants, although in practice it's nearer 30 - 50%. Either way, the proportion of recombinants is high enough to allow you to screen for insert directly.

Hope this helps

Andrew

In one case I actually found that the correct orientation gave me colonies that were blue - although slightly lighter. It turns out my insert was in frame and the fusion protein still expressed the LacZ gene.