Welcome to the Tol2kit blog

This is a forum for discussing the Tol2kit; the Lawson lab's related system; and related topics.

The Tol2kit is a set of constructs that can be used with Invitrogen's multisite Gateway recombination-based cloning system to build expression constructs in a Tol2 transposon backbone (derived from constructs from the Kawakami lab).

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Wednesday, February 7, 2007

Report an error

If anyone finds errors in the documentation, sequences, or (god forbid) plasmid spotting, please report them here.

3 comments:

Tom Hall, Currie Lab said...

Hi, I find an extra KpnI site in the pDEST-Tol2-pA2 and pDEST-Tol2-CG2 vectors.

Digesting with EcoRI gives a single band of 5877 for pDEST-Tol2-pA2 and a band of 7790 for pDEST-Tol2-CG2.

Doing a double digest with EcoRI+KpnI gives approximate sizes of 500+5377 for pDEST-Tol2-pA2 and 500+7290 for pDEST-Tol2-CG2.

jim lister said...

I too had a Kpn I site pop up unexpectedly when I was screening some clones from an LR reaction. I was using a Destination vector I'd made myself with a ClaI-XhoI fragment from pDest-Tol2pCG that contained the R4R3 gate. Originally I thought the KpnI site was just something I'd overlooked in the backbone of my vector, but now it would appear from Tom's digests and those that I did that there is a Kpn I site somewhere between the attR3 site and the ClaI site?

Chi-Bin Chien said...

Thanks Tom and Jim. We have tracked down the problem: there is indeed an extra KpnI site just next to the ClaI site. This was in the original pDestTol2pA vector made by Clemens Grabher, and thus is in all three derivative vectors (pDestTol2pA2, pDestTol2CG, pDestTol2CG2). We will fix up the sequences and post the correct ones ASAP.

Hope this didn't mess up anyone's experiments; this site does not affect the functionality of any of the destination vectors.