A multisite Gateway-based construction kit for Tol2 expression constructs
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, April 25, 2007
Wish list and new constructs
If anyone has requests for constructs that they'd like to see, or has made clones that they'd like to share, comment below.
Something equivalent to p5E-MCS or p5E-FseI-AscI for the middle and 3' entry vectors would be handy, to provide an alternative to BP cloning for making those components (although it may make things tricky when it comes to in-frame fusions).
Along these lines, we have made a middle entry clone with most of a CS2 MCS that we are testing now, with the idea that ORFs one already has in CS2 could be easily swapped over by conventional cloning.
We haven't done anything with EBFP, and we don't know of anyone else who has, so if you would like to make some clones, that sounds great!
I have just made middle clones of EGFP and mCherry without stop codons to do exactly what you've suggested; once they're sequenced and verified to work, we can include them in future Tol2kit distributions.
The H2A- and -CAAX fusions were I believe all made by fusion PCR. We have been using Phusion polymerase (NEB) which has been giving the fewest PCR errors in our hands.
The constructs in the initial release of the Tol2kit (Jan 2007) were built primarily by Kristen Kwan and Esther Fujimoto in the Chien lab, with contributions from Chi-Bin Chien, Melissa Hardy, and Ben Mangum. The pDestTol2pA destination vector was built by Clemens Grabher in Tom Look's lab, based on the Tol2 transposon originally isolated and described by Koichi Kawakami. Many thanks to Nathan Lawson and Jochen Wittbrodt for their extensive help and advice.
8 comments:
I'll start: We are thinking of making a 5' clone with the following insert:
FseI-AscI-basal promoter
The idea would be that you can PCR up your enhancer fragment with FseI-AscI ends, then clone it in conventionally to give:
FseI-enhancer-AscI-basal promoter
It'd be a while before this is made though. Any volunteers?
Hi Chi-Bin,
Something equivalent to p5E-MCS or p5E-FseI-AscI for the middle and 3' entry vectors would be handy, to provide an alternative to BP cloning for making those components (although it may make things tricky when it comes to in-frame fusions).
Along these lines, we have made a middle entry clone with most of a CS2 MCS that we are testing now, with the idea that ORFs one already has in CS2 could be easily swapped over by conventional cloning.
-Jim
Hi, there is new BFP (EBFP2) from Robert Campbell's lab which can be used in conjuction with both GFP and mCherry (see Biochemistry 46: 5904 - 5910)
I am thinking of making some clones unless someone has already done it?
Hi - another question, have you thought about making pME-EGFP and pME-mCherry without stop codons to allow N-terminal fusions?
Hi Tom,
We haven't done anything with EBFP, and we don't know of anyone else who has, so if you would like to make some clones, that sounds great!
I have just made middle clones of EGFP and mCherry without stop codons to do exactly what you've suggested; once they're sequenced and verified to work, we can include them in future Tol2kit distributions.
Best,
Kristen
Thanks Kristen,
I was wondering what strategy you use for making the H2A and caax fusions. I can't seem to find convenient restriction sites!
Tom
Hi Tom,
The H2A- and -CAAX fusions were I believe all made by fusion PCR. We have been using Phusion polymerase (NEB) which has been giving the fewest PCR errors in our hands.
Fantastic!
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