Big cloning

[ANDY PHILLIPS]

As regards cloning of large fragments into plasmids, many years ago I cloned 
a set of chloroplast DNA fragments into pUC8: about half of these were over 5kb 
and several were 18-22 kb in size. I did this by ligating PstI digested 
chloroplast DNA to CIP-treated, cut vector. I picked about 50 random clones to 
identify the smaller clones and then used colony hybridization with labelled, 
isolated fragments to identify those with larger inserts, which were much 
rarer. I used the Hanahan hexaminecobaltchloride/DTT method to increase 
transformation rate.

I suspect that the key part of the above strategy is that the insert DNA has 
never been down an agarose gel. Isolating a large fragment is especially 
tricky, as it is more likely to pick up UV damage on the transilluminator than 
is a small fragment. Maybe methylene blue staining would help here, but there's 
still the problem of contaminating agarose and impurities carried through 
purification. You don't need that when you're pushing your luck anyway.

You should probably also use a recA- strain for the cloning, as larger 
fragments have more chance of containing troublesome sequences. Having said 
that, I used JM83 (which I think is rec+), but there was a single chloroplast 
DNA fragment that refuse to clone.

We've also found recently that although electroporation can help in cloning 
large fragments, as it increases the efficiency of transformation, not all 
electroporators appear to be equal. We spent some time trying to use to clone 
a 6kbp fragment into an 8kbp transfer vector with a cheapo electroporator 
(Invitrogen) with no success. When we used the (expensive) all-singing, 
all-dancing BioRad machine, it worked first time. This may be a function of the 
specific plasmid or insert, but it suggests that not all electroporators are 
created equal. However, the Invitrogen machine is fine for most things.

Cheers,

Andy

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