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. Please post with your full name and institution (not just your first name or "Anonymous"), as it is helpful for other readers to know who is asking.

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Wednesday, January 31, 2007

Questions on Lawson lab Gateway constructs

Ask questions about the Lawson lab constructs by commenting on this post.

10 comments:

Anonymous said...

Dr. Chien,Can you put the maps of entry plasmids 228 p5E-MCS and 381 p5E-Fse-Asc on the website so that we can change the MCS for our own entry plasmids? Thanks.

Chi-Bin Chien said...

Hi there,

Sorry about the delay in getting sequences and maps up, other tasks are intervening.

p5E-MCS contains the Bluescript MCS, from T7 primer through T3 primer.

p5E-Fse-Asc only has an Fse site, then an Asc site.

I am posting on the wiki the sequences (most useful) and crude maps (not restriction maps, unfortunately) of these two constructs.

In the future it would be most useful to NOT comment anonymously...

George Zanazzi said...

Dear Dr. Chien,

Have you made an entry vector analogous to your p5E-MCS, but with the MCS in the T3->T7 orientation? If not, are you aware of anyone who has done this?

Thank you,
George Zanazzi

Chi-Bin Chien said...

Hi George,

Nope, we have not made a 5' entry vector with a different multiple cloning site. The only related construct we made was p5E-Fse-Asc, which only has two 8-cutters. It would be very easy to make such a construct though.

Also please note that this thread is meant for the *Lawson lab* system. There is a separate thread for our Tol2kit system.

Kate Lewis said...

Hi - we would like to try and use this system for promoter analysis. If I am understanding things correctly - to do this we could insert our potential promoter sequence into construct 213 and then do a multisite reaction with this alterred construct and constructs 353 and 465.
Is this correct?

If so - would it be possible for us to obtain constructs 465 and 353 from you? (Unfortunately they weren't on the wonderful sheets of plasmids handed out at Asilomar)

As far as I can tell - there isn't a way to do this with the vectors from Chi-bin's lab - is this correct?

(I'm new to all of this so I very well might be missing something)

Thanks for all of your help with this

All the best
Kate

jim lister said...

Hi Kate,

I haven't used any of Nathan's constructs but I believe you're correct, if it's a potential *enhancer* that you want to check (i.e. if it is likely to lack its own basal promoter). However if you have a putative *promoter* (including sequence up to the transcriptional start site) you can still use the Tol2kit for that. Or if you just want to do a simple Gateway reaction I've made a couple of plasmids that have a single gate (with no basal promoter) 5' of EGFP/SV40polyA. Unfortunately my constructs require that your promoter be in an entry vector with an L1-L2 gate rather than L4-R1 so they aren't compatible with the other Lawson or Tol2kit plasmids. On the other hand, the single Gateway reaction works very efficiently, and Invitrogen sells a TOPO cloning kit with an L1-L2 gate in the vector so you can generate your promoter by PCR and subclone it without having to add att sites to your primers and then do a BP reaction (although you do have to add a particular 4-base sequence to your upstream primer for the directional TOPO cloning reaction).
Let me know if you want more info.
-Jim

jim lister said...

Now that I've looked more closely at the Lawson lab page, I see you can set things up with that system to switch promoters in and out using single Gateway reactions as well (and be able to use it for other multi-Gateway applications once you've found a promoter that's suitable to your needs.)

Chi-Bin Chien said...

Kate, Jim Listers' comments are right. You have different options depending on whether you are characterizing a promoter or an enhancer.

For promoters, you can use the Tol2kit. Just clone them into a p5E clone, and combine with pME-EGFP, p3E-polyA, and pDestTol2pA2.

For enhancers, use instead Nathan's 353 pENTRbasEGFP, which has its own basal promoter. I believe that this was in the set of plasmids that he gave out at Asilomar.

It will seem awkward using multisite Gateway for these experiments, but the payoff is that once you have an element that you like, you can immediately make Cherry versions and nls or membrane-localized versions.

Monika Gostic said...

Hi,

I;m performing a multisite recombination and am recombining p5E-MCS (L4/R1) and pME-mCherry (L1/L2). Could you tell me which enzyme to use for this recombination? BP clonase? LR clonase? Many thanks!

Monika Gostic said...

Hi,

I;m performing a multisite recombination and am recombining p5E-MCS (L4/R1) and pME-mCherry (L1/L2). Could you tell me which enzyme to use for this recombination? BP clonase? LR clonase? Many thanks!