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).
At present each post serves as a topic under which visitors may post comments, either commenting or asking questions. We encourage you to post with your full name (not just your first name) so that we have some idea who is asking.
30 October 2007: There has been a fair amount of spam recently, so I have turned on word verification for comments. You will have to do a "word recognition" task before you can post a comment. Please email me directly if you have any problems with this. --Chi-Bin
3 comments:
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.
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?
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.
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