Friday, April 12, 2013
Questions on the Tol2kit, 4/12/13 onwards
Ask general questions about the Tol2kit system by commenting on this post; we will do our best to answer in a timely manner. Reminder: please use your full name and institution, so that we know who is in the conversation. This posting covers questions from 4/12/13 onwards.
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35 comments:
Does anyone has tried to electroporate a Tol2 construct with a Transposase plasmid such as pCAGGS-Transposase.
It has been described in chicken but not in fish.
Any advice would be great!
Thanks,
Jonathan Boulanger-Weill
German Sumbre Lab
IBENS
Hi Kristen,
I'm Erin Booton from Josh Gamse's lab at Vanderbilt. Thank you so much for all your help and your work on this blog. I have a simple question about the middle entry clones containing Gal4 or Gal4FF (#676, #705, and #781) from the 2.0 version of the Tol2kit. Do they contain stop codons? I appreciate your help so much!
Erin
Dear Erin,
All three of those clones have stop codons. Hope this helps!
Best,
Kristen
Thanks Kristen that is good to know.
Erin
Hi,
I'm Mike at Northeastern University. I have an expression clone in the pTolDestR4-R2pA vector. This may be a gateway question, but I would like to switch out GFP for mCherry. Is it possible to do a BP reaction with a donor vector (containing ccdb/CmR and attP sites) to revert it back to a destination vector? Then do an LR reaction to insert mCherry?
Thanks so much for your help!
Mike
Hello,
I have a question: I would like to design reverse primers over the AttB3 site (multisite cloning) for sequencing, to ensure that our 3'element is integrated. Has anyone tried this? Does anyone happen to have a sequence that works?
Hello,
I'm Kaj from Sloan Kettering. I have a question: I would like to design reverse primers over the AttB3 site (multisite cloning) for sequencing, to ensure that our 3'element is integrated. Has anyone tried this? Does anyone happen to have a sequence that works?
Dear Mike,
In theory, the "reverse BP" is possible; I have heard of cases of people doing such a reaction. We have not done one ourselves. If you try it, just be sure to do the double selection (with chloramphenicol) and use ccdB- cells.
Good luck!
Kristen
Dear Kaj,
We have not done this ourselves; it is probably possible to generate a reverse primer over the attB3 itself? My only concern is that if the recombination has not occurred correctly, the attB3 sequence might not be there. You might want to check the sequence just downstream of the attR3 site in the destination vector.
Hope this helps,
Kristen
Thanks for your help Kristen!
Mike
This is cool!
Hi everyone,
my name is Andreas Brodehl (University of Calgary). We want to express an construct only in the heart of zebrafishs. Is there a specific promotor construct (like MYH7 promotor) available? Thank you very much
Dear Andreas,
Currently many of us use the cmlc2 (myl7) promoter. It is included in the Tol2kit within the pDestTol2CG2 plasmid as a transgenesis marker (driving EGFP). If you have access to that plasmid, you should be able to PCR it and clone it into whatever form you need.
I hope this helps,
Best,
Kristen
Hi there
I have 2 questions:
1. I have have done transformation using kanamycin plate with a concentration of 50mg/ml and now I want to grow them for pDNA extraction. So do I need to reduce the kanamycin concentration to half (25mg/ml) or simply use the same concentration.
2. There are certain strains that do not show colonies after transformation with kanamycin plates (as recommended)the concnetration I use is 50mg/ml...therefore do I need to decrease the amount to half of its original contration???
Hi there
I have 2 questions:
1. I have have done transformation using kanamycin plate with a concentration of 50mg/ml and now I want to grow them for pDNA extraction. So do I need to reduce the kanamycin concentration to half (25mg/ml) or simply use the same concentration.
2. There are certain strains that do not show colonies after transformation with kanamycin plates (as recommended)the concnetration I use is 50mg/ml...therefore do I need to decrease the amount to half of its original concentration???
Regards
Thomas (CUHK)
Hello,
I'm Jenny Rahn from Sherine Chan's lab at the Medical University of South Carolina. I have been bringing clones up from the filter paper (it was stored at -80) and have had trouble with #237 (pME-MCS) and #219 (pDONRP4-P1R). All the other clones I have tried to bring out have worked without any problems. I tried switching the plating/transforming conditions between these two in case I mislabeled the spots but that didn't work either. I have tried several E.coli strains for the 237 and have tried several amounts of plasmid in the transformation for the 219 but have not obtained any colonies. Is there anything you can think of to try or is there any way I might be able to get another spot of these two plasmids? Thanks so much!
Jenny
Hi there, I'm Monika from St Andrews Uni, working on Zebrafish neurogenetics. I was wondering if there is any construct with RFP instead of GFP or mCherry? Maybe Mike from Northeastern University knows how to do it (did you manage to switch them?). Any suggestions are welcome! Thank you!
Hi Monika,
The Lawson lab made a DsRed2 entry vector, and the latest update of the Tol2kit (2.0) has some vectors with mApple.
Jim
Hello everyone
I'd like to know if anyone has made a middle vector GAL4FF without stop codon in order to use 2A-mcherry in a 3'vector.
I'd really appreciate some help about it!
Thanks in advance
Leo!
Hello!
I'm Anna Krasnow from Steve's Wilson lab at University College London. Following up from Erin's question - given that all the Gal4 middle entry clones from the 2.0 version of the Tol2kit have stop codons, I was wondering if anyone has generated such a clone without a stop codon? This would allow one to make a 'gal4-2a-fluorescent protein' constructs, making the kit so much more versatile and useful!
I would welcome any comments/help, thank you!
Kind regards,
Anna
This is Brent Bill from UCLA. I just noticed that the main tol2 wiki previously curated by the Chien lab (http://chien.neuro.utah.edu/tol2kitwiki/) is offline. Is this a permanent removal? Is there an attempt to move this information to the zfin protocols wiki?
Or is there an updated site that would be better to access?
Thanks,
Brent
Dear Brent,
I apologize for the lapse -- we have migrated the wiki to a new website, and are waiting for the university to allow us to redirect traffic from the old Chien Lab site to the new site; our request has already been submitted.
Here is the new Tol2kit wiki site: http://tol2kit.genetics.utah.edu
Please let me know if you have any trouble. We hope to have the old URL active to redirect soon - thanks again for your patience.
Best,
Kristen
Hi Kristen,
I am a postdoc in Rich White’s lab at MSKCC. I have a question about Tol2 cloning. We are trying to create a new 395 gateway backbone with a tdTomato marker gene (basically swapped out the cmlc2-gfp in #395) that we can then use for standard three way gateway. To test it, we gateway’d in a ubiquitous-CFP. The marker in the backbone is oriented opposite to the gateway insert clone. So, the fish should be both red and blue. However, it seems that the two genes are interfering with each other – fish that are red aren’t blue, and fish that are blue aren’t red.
I know that this can happen on large plasmids (12kb for the new 395 backbone, 16kb for the final expression vector), but do you have any suggestions on how to improve this? Would miniTol as a backbone be any better? Or insulators of some type?
Thanks for any help!
Yan Zhang
Hi All
I am looking for a 5' entry plasmid with the Fli enhancer. I know that there is a super short version, however is there a more complete version?
Thanks a lot
Liron Gibbs-Bar
Weizmann Institute for Science
Hi,
I was wondering what is the size limit of the gene the Tol2 system is capable of delivering into the zebrafish genome. My gene of interest has a considerable size and I am not sure whether Tol2 would work for big chunk of DNA. Thanks!
Hi, I'm currently trying to make an N-terminal eGFP fusion and have been having a few problems. I was wondering if anyone has a protocol for how to do so?
many thanks
Andy Nichols
Qwarnstrom Lab
University of Sheffield.
Hi,
This is Katherine Rodgers from the Scott lab at the University of Florida. We have received from another lab the pCS-TP and modified oT2kXIGdeltain plasmid with an inserted tissue-specific promoter. Our lab transformed some DH5alpha E.coli with the plasmids but are having trouble confirming with restriction digests after minipreps. The plasmid size is too small and not digesting as we expected. We are wondering if there is another type of E.coli we were supposed to use with this system? Our theory was that maybe there was a recombination event in the oT2kXIGdeltain plasmid that released the EGFP and promoter. Can you give any advise?
Thanks.
Sorry if this is obvious or covered elsewhere - I haven't found it yet within the blog - but are the sequences for the v1.2 kit available on Genbank? There are postings that say all sequences are uploaded, but I can't find any of them. Maybe that's just my inability to navigate around NCBI, but if anyone has any tricks or tags that can help pull them up, that'd be great.
John Willoughby
UMASS Amherst
Hi John,
The sequences are available in GenBank and FASTA formats here:
http://tol2kit.genetics.utah.edu/index.php/List_of_entry_and_destination_vectors
There aren't any sequences for the v2.0 kit though, you may have to ask Kristen to send you those (or if you're looking for particular sequences, I'm happy to send them to you -- I think I probably have most if not all.)
Jim Lister
VCU
Many thanks!
I had meant to add that yes, many are version 2.0.
I have sequenced into the couple that I need immediately, but will perhaps ask you in future for version 2.0 sequences if needed. Thanks for that kind offer.
John
Hi Everyone,
Paul Wrighton from Wolfram Goessling's Lab at Brigham and Women's Hospital. I'm also hoping to find sequences for some of the plasmids in version 2.0. Are these available online anywhere? If not, I'm especially interested in finding the sequence for p5E-nlsmCherry (766), but also: p5E-MCS polyA (633) and pDestTol2pA3 (514).
Thanks,
Paul
Hello,
This is Lana Pollock from the Anand-Apte lab at the Cleveland Clinic. I noticed a possible error in the "Att site sequences" page. The attB2 and attB2R primer sequences seem to be reversed compared to those listed in the Invitrogen manual.
Thanks,
Lana
Dear Lana,
This depends on which Invitrogen Manual you are using - our primers and nomenclature are specific for the older version Gateway (3-part MultiSite), and I realized (thanks to your comment) that the link to the old version PDF is broken on the wiki.
I've now updated the link on the wiki, and the primer sequences are listed on p. 25 of the PDF (p. 15 of the manual).
Hope this helps,
Kristen
Hi everyone,
I'm Charlotte and i have to construct several transgenics lines. I'm missing the pDESTTol2pA (392) and the pDONRP2R-P3 (220). Is anyone have these plasmids in the lab'?
Thanks!
Charlotte
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