Hi Geir,

Have you check for lytic phage contamination?

Use a lytic resistant E. coli strain instead of xl1-blue.

Fred

could you have a look at the papers from Ronald Kaback group?

they are very halpfull.

Sometimes you need a new and fresh CJ 236.You can look as well at my paper..

On the ohter hand ,Maniatis has a good protocol.

Hi Geir,

I suspect that your helper phage stock is contaminated with another phage(mid), which is a common problem when one simply amplifies the helper phage by infecting E. coli. I'd suggest single plaque isolation of the helper phage using the soft agar method, followed by phage propagation.

Shohei

Hi,

I am agree with Shohei for his comment about Phage stock contamination, please check it.

good luck

Thanks Shohei,

We've looked into helper issue due to the known problem, as you mentioned. Hence, we could reliably rule it out. The issue has now been circumvented by adopting the protocol to a CJ236 independent flow. The fusion is toxic, hence I speculate in that it could be a recA-mediated effect that is clone dependent. Have never seen it before myself, nor seen it reported though.

Hi Geir, I experienced problems with isolating ssDNA but most of the time if I used fresh stock of the helper phage helped out to solve the problem.

We met the exactly same problem too.  Sometimes the protocol works well, sometimes not... maybe it is really  clone dependent. I m trying to figure it out.