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.

To help prevent spam, word verification is turned on: you will have to do a "word recognition" task before you can post a comment.

Tuesday, April 3, 2012

Questions on the Tol2kit, 4/4/12-4/12/13

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 4/4/12 onwards.

51 comments:

Unknown said...

Hi I am Gianluca from Hazel Sive lab. I made several Tol2 constructs with different promoters. when I digested them with different restriction enzyme I found out additional fragments, sometime they look like as kind of extra non-digested DNA. is it normal? moreover when I injected them at a concentration of 30 ng/ul with 25 ng/ul of transposase I got an high percentage of death/monster embryos. can you help me to figure out where is (if there is) the problem?
thanks
Gianluca

Kristen Kwan said...

Hi Gianluca,
First, with respect to your constructs, having additional fragments upon digest is not normal. I would be very hesitant to use such constructs. If this has been a consistent problem independent of the entry vectors you use, you should perform a test digest of your destination vectors to make sure that they are OK. This sounds to me that perhaps something is off with your destination vector.
Second, with respect to your injections, your concentrations seem OK, but just to be clear, we inject 1 nl of those concentrations for a total of 30 pg DNA and 25 pg transposase RNA. Are you doing the same? A significant number of dead/deformed embryos is normal; we expect ~50%, as a sign that the transposase is active and transposition is occurring.
Hope this helps, and let me know if you have further questions,
Kristen

Gianluca said...

Dear Kristen
I double checked once again all my entry and destination clones with same digestion enzymes I used for the final clones and everything looks perfectly normal. I don't understand why I am not able to digest normally my final constructs.
about my injections, I inject 1nl and I have also nice expression respect to my promoters but since I would like to do also an F0 analysis I was wondering if the death/monster percentage I got was normal.

thank you again
best
Gianluca

Anonymous said...

Hello,
My name is Zachary Gaber, a grad student in the Novitch Lab at UCLA. I posted a question a few weeks ago and I'm still more or less hung up on the same step. I can't get a successful BP clonase products into pDONR-P4-P1R. Part of the snags have been in unrelated matters (amplifying a tough template, a problem with our competent bacteria - something pink kept growing on the plates) but now I'm really caught on the pDONR-P4-P1R BP clonase reaction. I've done literally dozens of successful BP clonase reactions into the middle and 3' elements, but I can't get this one to work.

You mentioned that pDONR-P4-P1R undergoes spontaneous recombination and maybe that is the problem. We weren't aware of this problem when we made our prep of this vector, so we took no precautions against it. However, I've been leery of spending the time to make a new prep as my problem doesn't sound like what I would expect self-recombination would look like. I would predict that if self-recombination was occurring in my BP clonase reaction, there would be many hundreds or even thousands of background colonies that don't possess either the insert or the CmR/ccdB cassette but would still have the pDONR sequencing sites. That's not what I'm getting. I'm getting perhaps a few dozen background colonies (or often none at all) and when the background colonies are sequenced, the results come back "no signal". Presumably because whatever is in those colonies, it doesn't have M13 sites.

Am I wrong in thinking that a bad prep of pDONR-P4-P1R should produce hundreds or thousands of colonies? Am I wrong in thinking that the spontaneous recombination would preserve M13 sequencing sites? What does a bad prep look like after a BP reaction?

Any suggestions would be appreciated... right now all I can think of is making a new prep of pDONR-P4-P1R even though I don't see how that could be the problem or maybe just using restriction enzymes to clone into p5E-MCS, although admittedly that's not a good longterm solution. Thanks.

Kristen Kwan said...

Dear Gianluca,
It's good that your digests look OK. But again, if this is something that you see consistently regardless of which entry clones you use, I would still be a bit concerned about the destination vector. It might be worth trying a couple of other diagnostic digests (different enzymes) or sequencing the R4/R3 ends.
In terms of doing F0 analysis, just a tip: when we do this, we often try 2-3 different concentrations of plasmid to get the "right" percentage of monsters (~50%). Since we have generally measured plasmid amounts in pg, the actual molar amounts can vary wildly depending on the size of the plasmid.
Good luck!
Kristen

Kristen Kwan said...

Dear Zachary,
In our experience, a bad prep of pDONR-P4-P1R gives hundreds of colonies. To be honest, we haven't sequenced them, so I'm not entirely sure what's left after the self-recombination.
Is there someone else around UCLA from whom you can get a bit of trustworthy donor vector? If you can give us a few days (lab is in transition right now), we could also send you some. Email me (kristen.kwan@neuro.utah.edu).
One other thought occurred to me: how big is the enhancer you are trying to clone? If it's very large (>12 kb), we take some particular precautions (use Electrocomp cells, grow at 30 or 32 degrees).
Let me know what you think -
Kristen

chunlin said...

Dear Zachary,
This is Chunlin at TLL singapore, I've got similar experience with you when I worked with pDONR-p4-p1r. But the problem is solved now.
pDONR-P4-P1R undergoes spontaneous recombination, while it seems not between p4 and p1r to remove the ccb gene, since I never got any clonies growing on my plates when I used a bad pDONR-P4-P1R (same as you).
And this spontaneous recombination might be happened with your good preparation during the storage in -20 degree. I tried to retransfer my good pDONR-P4-P1R to ccb compatent cells, I picked 4 clonies for mini-prep and sequencing by M13F primer, all give reads, but only one is correct.
Therefore, the solution for you is 1,get some good pDONR-P4-P1R; 2,try to retransfer your original one, pick more single clony, and get the good one.
Hope this may help.

Jean said...

Hi,
My name is Giacomotto Jean. I am working with the Gal4/UAS system, and I am wondering if someone has already work with an engineering P5e-UAS construct, like presented in Courtney et al, 2011 ? -In aiming to minimise methylation-. Does that work well for you ? Enough expression in your studies ? Over multiple generations ?
Cheers all,
@J

Anonymous said...

Hello,
My name is Robert from the Neuhauss lab. I made several Tol2 constructs with different promoters driving GalVP16. After injection into our reporterline I screened for fishes that transiently and correctly expressed the reporter and raised these to adulthood. Then I screened the grown up F0 fishes and found several F1 founder fishes (F0 positiv with positive progeny).
Now my problem: In the F1 founder progeny I get (not frequently, but at least always one) larvae that expresses the transgenesis marker but shows no reporter gene expression. To me this makes no sense, since if silencing would occur then the whole construct would be silenced, am I right?

It is actually not a too big of a problem since I just don't take these to establish my transgenic lines, but I was just wondering and coulnd't make much sense out it...
Thank you for your help,

Sincerly,
Rob

Kristen Kwan said...

Hi Rob,

So my first thought is that the silencing is really affecting your UAS transgene, and not necessarily your Gal4VP16 transgene. Do you know for sure that the Gal4VP16 is being silenced, i.e., that Gal4VP16 protein is lost? Have you done an in situ or antibody staining for Gal4VP16?

UAS transgenes are notorious for being susceptible to silencing, so it would not be surprising if that was where your loss of expression was occurring.

What do you think?
Kristen

Anonymous said...

Hi again,

First of all: So far I did not perform an in situ / antibody staining for Gal4VP16 but your suggested UAS transgene silencing makes much sense to me.
Anyway, allthough annoying, it is not too big of a problem for me. I just dont raise these larva and everybody is fine :)

Thanks for help,
Rob

greengirl1808 said...

Hi my name is Amber,
I have been trying to design PCR, and eventually qPCR, primers for Tol to determine the number of insertions in my transgenics (without using Southern blotting). I have read a lot of the Kawakami lab papers to try to determine what is the Tol sequence after insertion? Can anyone refer me to a paper with this information or tell me the sequence?
Thanks so much!

chiara milanese said...

Hello everybody,
I am Chiara from the ErasmusMC University. I'm currently using the Tol2 system to express a fluorescent fusion protein. I am able to see the right expression only at 24-48 hours but then it disappears from the embryo. When I genotype the embryo that was previously expressing it I do find the insert at 6dpf, but no fluorescence. Do you have any idea of why is it happening?
Thank you
Chiara

rocky said...

Hi, I'm Rakesh from Glasgow Caledonian University. I have a question about the primer attB design, is it necessary to include two additional nucleotides in the forward primer? I have been struggling to get a successful BP reaction. Could anyone please help me to figure it out?
Thanks
Rakesh

Kristen Kwan said...

Dear Chiara,
It sounds to me like your promoter is the issue. Is it possible that it stops driving transcription after 24-48 hpf?
Kristen

Kristen Kwan said...

Dear Rocky,

It is not absolutely necessary to include the two additional nucleotides. We do out of habit, just to make sure everything is standardized. It is most critical in cases where you want to keep different entry clones in the same translational reading frame.

Are you confident as to the quality of your donor vector? How big is your insert? Which entry clone are you trying to make? We note on our Tol2kit wiki that the 5' donor vector (pDONR P4-P1R) is prone to self-recombination. Nathan Lawson's lab has a protocol for propagating that vector. Also, if you haven't, please take a look at the Protocols page on our wiki for some tips regarding BP reactions.

rocky said...

Dear Kristen,
Thanks a lot for the reply.
I have followed Lawson's lab for propagating the pDONRP4-P1R (used chemical competent cell) and I am not getting any colonies for the BP reaction using pDONR221 either.

The inserts for the entry clone are 3Kb. I am using pDONRP4-P1R and pDONR221 for the entry clones and will be using pDESTTol2CG2 as destination vector.

Thanks
Rakesh

Kristen Kwan said...

Dear Rakesh,

I assume you used ccdB- cells to propagate your donor vector? Other than this, I would make sure that your primer sequences are correct (I have seen multiple instances of failed BPs due to using, for example, AttB1F and AttB2F for recombination into pDONR221), and then worry about your clonase - has it previously worked?

Kristen

rocky said...
This comment has been removed by the author.
rocky said...

Dear Kristen,
Yes, I have used ccdB cells (home-made)for propagating thr donor vectors. The primers I am using are
pDONRP4-P1R
Forward attB4:GGGGACAACTTTGTATAGAAAAGTTG-template specific
Reverse attB1:GGGGACTGCTTTTTTGTACAAACTTG-template specific
pDONR221
Forward attB1:GGGGACAAGTTTGTACAAAAAAGCAGGCT-Nflag
attB2:GGGGACCACTTTGTACAAGAAAGCTGGG-template specific

It did worked once, sequence using M13Forward primer showed attB sequence of the PCR product into the PDONR221 (plasmid from the colony picked from a LB plate (no antibiotics used)).
I got new the clonase and the perform the BP reaction strictly following the multisite gateway protocol.

I use Expand Long template PCR system from Roche - (mix of Taq and tgo DNA ploymerase) is there is any chance that affect the att sites?


Sorry for the trouble!

Thanks
Rakesh

Kristen Kwan said...

Dear Rakesh,

Your primers look fine. But i'm confused about your experiment that worked. Why did you transform onto a plate with no antibiotic? Also, after recombination, that Att site should be an L site, not a B site. Are you sure that recombination occurred correctly?

Have you also looked at the protocols on our Tol2kit wiki? We have found that putting the purified PCR product through a freeze-thaw cycle extremely severely hampers the reaction. We don't know why, but as a result, we never freeze a purified PCR product before going into a BP.

Finally, I don't know if the polymerase would have an effect. We don't use that one; for templates on the order of 3 kb, we've had fine luck with Platinum Taq or Phusion.

Kristen

rocky said...

Dear Kristen,
Sorry , I got attL1 when I sequenced using the M13 primer and rest of the sequence matched the template.
I didn't get any colonies when I used LB plates with Kanamycin, so I tried without Kanamycin (just tried my luck picking a right one).

I will try to produce attB PCR products using Platinum Taq or Phusion.

Thanks Kirsten
Rakesh

Lobo said...

Hi everybody,

I am Thomas from the Friedrich lab in Basel and I wanted to re-post a question that I had posted earlier concerning the use of 2A sequences in the Tol2 kit:

It has been mentioned on this forum that people have (succesfully) used 2A constructs for bicistronic expression - rather than IRESs. Has anybody used a middle entry clone with a 2A sequence (e.g. Provost et al., 2007) (and obviously no STOP codon), followed by a 3' entry clone such as YFP? And if so, what would be your general impression of the 'danger' that (a) the extra C-terminal amino acids encoded by the 2A sequence and/or (b) the extra N-terminal amino acids of the 2A sequence and the recombined att site adversely affect the function of the 'middle' and/or '3'' protein, respectively? Obviously this might depend strongly on the actual proteins, but any comment about general trends or succesful examples would be very helpful. In my hands it has worked nicely in some cases (e.g. KillerRed-2A-EGFP) but not in others (e.g. channelrhodopsin-2A-halorhodopsin)

Many thanks in advance!

Cheers,
Thomas

Kristen Kwan said...

Dear Thomas,

We have used the 2A peptide for biscistronic expression. But the way we have designed it, we have a gene of interest (with no stop codon) in the middle position, and a 3' clone containing the 2A peptide followed immediately by an FP. In our situation, the middle clone has a long C-terminal fusion (7 aa from Att site + 21 aa from 2A peptide), and the FP is preceded by a single extra aa (proline).

Our experience has been exactly as you say - it depends very strongly on the protein of interest. In cases where the protein of interest receives a C-terminal modification, we've had to reverse the genes and put the FP in the middle position, with the 3' clone containing the 2A peptide followed by the gene of interest.

In your case where channelrhodopsin-2A-halorhodopsin didn't work, that could be because of the extra amino acids on the front of your halorhodopsin. It has been shown that the single extra proline from the 2A peptide at the front of a secreted protein does not interfere with signal peptide recognition; I believe the entire T-cell receptor complex was reconstituted using 2A peptides. However, the way that you've designed your construct, the halorhodopsin has the proline + the 7 aa's from the Att site. That might interfere with signal sequence recognition.

Does that make any sense?
Best,
Kristen

Thomas said...

Dear Kristen,

thank you for your quick answer! It does make a lot of sense. And thanks for the idea of inverting the sequence of the proteins.

One last question: which 2A sequence(s) have you been using - PTV1, TaV, FMDV, or others?

Once again, thank you very much.

Best,
Thomas

Kristen Kwan said...

Dear Thomas,

We have only used the PTV1 2A peptide; we got it directly from Elayne Provost and Steven Leach.

Best,
Kristen

Thomas said...

Dear Kristen,

thanks. I really appreciate your efforts on the Tol2kit & the blog! Great job.

Best,
Thomas

emilydon said...

Hello,

I'm Emily Don from Nicholas Cole's lab at the University of Sydney. Does anyone have the sequence for pDONR P4-P1R? I cannot find it anywhere. If possible, could it be emailed to me at emilydon@anatomy.usyd.edu.au

Thanks so much,
Emily

Anonymous said...

Hi,

I'm new to transgenesis and I'm afraid I have a very basic question.
I would like to make a hsp70-gene of interest-EGFP construct using the Tol2Kit. I was wondering if there are any reasons why I should preferentially use the genomic DNA rather than the cDNA of my gene of interest. It seems that in mice, expression of the gene of interest is more efficient with the genomic DNA, and when using cDNA people add an artificial intron to help getting the mRNA out of the nucleus. I couldn't find this kind of information for zebrafish. Are there the same type of considerations?
Thanks a lot in advance for your help.

Sophie Colombo (Columbia University, New York)

Kristen Kwan said...

Dear Emily,

I just emailed you a sequence file for pDONR P4-P1R. Please let me know if you have any questions or concerns.

Best,
Kristen

Kristen Kwan said...

Dear Sophie,

The short answer to your question is yes - the same kinds of concerns apply to zebrafish, although we haven't gotten far enough to incorporate introns in the Tol2kit as a standard practice. In our hands, we have not had trouble expressing cDNAs or fusion proteins that are < 3 kb. Once you get to ~3.5 kb, expression does appear to decrease. Incorporating introns into our Tol2kit has been on our to-do list for some time.

I will say that if you intend to use the hsp70l promoter that comes with the Tol2kit, it actually has a small intron. We have found that even with longer, hard-to-express constructs, the hsp70l promoter will induce expression (possibly both because it is very strong and has the intron).

Does this help?
Kristen

Anonymous said...

Dear Kristen,

Thanks a lot for your answer, this is very helpful.

Best,
Sophie

Anonymous said...

Hello,

I have cloned a PCR product into a TOPO vector and then into a tol2 vector. Is it possibe to clone the PCR product directly into tol2 and why?

Anonymous said...

Hi,
I wondered if there was an entry vector (3' element) with IRES driving a red fluorescent marker like RFP or mCherry?
Thanks
Emma

Kristen Kwan said...

Dear Emma,

The short answer is yes - we have the IRES driving mCherry (cytoplasmic) or TagRFP (cytoplasmic, membrane, or nuclear).

Here's the long, qualifying comment: we never distributed these for a couple of reasons. Regarding the IRES-mCherry construct, despite the sequence being correct, we could never see mCherry fluorescence, even by confocal. However, we have poor lasers here for visualizing mCherry, so we did send the clone to another lab (with a 561 laser line instead of 543), who said that they could see mCherry. So in theory, that clone should work fine and we could send it to you.

Regarding the TagRFP clones: TagRFP is under commercial MTA and we cannot send it to anyone unless they have bought the TagRFP clone from Evrogen. If you have done so, we can send it to you.

I hope this makes sense. Please email me at kristen.kwan@genetics.utah.edu if you would like me to send any plasmids to you.

Best,
Kristen

Anonymous said...

Dear Kristen,

Thanks for your reply it was really helpful. We haven't got the tagRFP clone as our lab doesn't have any experience with tagRFP. Does the IRES-tagRFP construct work well for you and therefore, in your opinion, would it be worth purchasing the clone?

Thanks for much for the advice
Emma

Kristen Kwan said...

Dear Emma,

It depends on your hardware and your needs. If you have a 561 or 594 laser on your confocal, you may be able to visualize the mCherry driven by the IRES, in which case you don't need the TagRFP clone. If you are saddled with a 543 nm laser like us, you may need TagRFP.

In our experience, TagRFP is bright and the IRES-TagRFP, although weak (as are all IRES-driven markers), it seems to work well enough. Two caveats: 1) despite its name "Tag"RFP and the fact that it was intended to be used in fluorescent protein fusions, we (and independently, another lab) had trouble making a fusion to a histone. The construct was lethal. Other fusions have been fine, however. So if you may want to use it to fluorescently tag a protein of interest in the future, just proceed with caution. 2) If you are concerned about speed of imaging, and you want to image both EGFP and TagRFP, you will likely need to perform sequential scanning on a laser scanning confocal, due to spectral overlap between the two fluorophores. This is not such a problem with EGFP and mCherry. So if you need fast imaging (for example, I do a lot of in vivo timelapse imaging), perhaps mCherry is a better bet for you (and does not require an MTA).

I hope this is helpful,
Kristen

Victor Li said...

Hi I am Victor from Marc Kirschner Lab at Harvard Medical School. I was wondering if the Tol2 system has any crossover with the PiggyBac system? That is, will the PiggyBac transposase excise Tol2 transposons and vice versa?

Kristen Kwan said...

Hi Victor,
Sorry for my delayed response. While I was fairly confident that there should not be any crosstalk between the PiggyBac and Tol2 transposases and transposons, I checked with someone who actually works on transposon biology. I am told that it would be unlikely and very surprising for there to be crosstalk - to the point that it would probably be a Science paper if it happened.
But just for the sake of being thorough, I would do the test to make sure.
I hope this is helpful - and please tell Marc hello. I don't know if you are aware, but I was a grad student in the lab. Hope everything is well there.
Best,
Kristen

Victor Li said...

Thanks, that's really good to know. I did know you were in the lab before...I think I saw you in that video you guys made for Marc's birthday :)

Cheers,
Victor

Lisa said...

Hi--I am Lisa from the Moens Lab, and we recently got the v2.0 kit from you. I was wondering when plasmid maps etc will be available?

Thanks!

Kristen Kwan said...

Hi Lisa,
It will be a little bit of time before maps and sequences will be posted on the website - the website has to be moved to a new server and migrated through several software upgrades (it's been a while since we really took care of it).
In the meantime, we obviously have sequence files. Email me (kristen.kwan@genetics.utah.edu) with the plasmid numbers of any plasmids you want files for right away, and I will email them to you.
Thanks for your patience!!
Kristen

Anonymous said...

Hi,

This is Mo from RBR Lab. We want to move some promoters (ranging 2kb-5kb) from p5E to pME, so we first att PCR up the inserts, and with the att-PCR products, we then do a BP reaction with pDONR221.

The BP reaction seemed to work well, as we got many colonies on the plate and they are the right size by digest, however, the subsequent LR hasn't been working.

Sequence results for the pDONR221 backbone and att-PCR products suggest both contain the correct att sites, but we found mutations at the att sites of the BP products, i.e. mutations at att sites after BP reaction.

Additionally, using the same approach (PCR->BP->LR), we successfully moved a coding gene from the p3E to pME, and subsequently generated the desired LR product.

Just wondering if you have ever come across difficulties cloning promoters into pME (since most promoters on the list are in p5E), and what you think went wrong in our approach to move inserts from p5E to pME.

Thank you.

Regards
Mo

Kristen Kwan said...

Dear Mo,

We have generally not needed to clone promoters into the middle entry clone, so I don't have a direct answer for your situation. From what you've told me, it sounds like a fluke result. So if I'm understanding you correctly, your empty pDONR221 plasmid is perfect, and the mutations only arise after BP reaction? And when putting your coding gene into the pME, you had no mutations?

The only time I've seen att site mutations that were induced by the BP reaction, it could be accounted for by mutations in the primers. If your primers are correct, I don't have any obvious reason for this to have happened. Did this happen for all of the promoters you tried?

Kristen

Anonymous said...

Dear Kristen,

Yes, the pDONR221 is perfect as shown by sequencing, and it was used to clone a coding gene from p3E to pME, successfully. Only when cloning promoters from p5E to pME we see att site mutations after BP.

To check the primers, we have cloned one PCR product (attB1-promoter-attB2) into pGEM and sent for sequencing, both attB1 and attB2 sites are what they should be. We have also ordered same primers from a different company, which give us the same errors after BP.

Yes, mutations in att sites after BP reaction happened in all three promoters we tried to clone into pME. They are done by different people in the lab as well!

We also don't understand why correct attB and attP will give incorrect attL. The only thing we haven't checked is the BP clonase II (invitrogen).

The plan is to borrow a working batch of pDONR221 and BP clonase from another lab, and test out whether it will give the same errors.

Thank you for the reply and will post after this trial.

Regards
Mo

Ursula said...

Hi,

I am Ursula a postdoc from the Laporte lab at IGBMC. We currently have huge problems using the multisite Gateway cloning reaction. Somehow we don't achieve successful recombination. We already ruled out many things: the Clonase is active as proven by a positive control reaction (4 plasmids that we successfully recombined before), we use Midiprep plasmid DNA, we even re-purified some plasmids and verified that the att sites are not mutated. Still we frequently don't manage to obtain the clones of interest. Has anybody an idea what may be the problem or what we could change additionally.I also wonder what kind of Midi prep kit you are using and if you also observed similar problems in some cases.

Thanks a lot in advance!
Ursula

Anonymous said...

Dear All,

I hope Kristen doesn't mind us posting this, and that it doesn't count as spam or advertising(!), but we've made a Tet-On toolkit comprising a few Gateway vectors which is available from us if anyone's interested.

(Many thanks to NL, CBC, KK for all of the vectors they've provided - this is our contribution)

John Willoughby (UMASS Amherst)

Campbell LJ, Willoughby JJ, Jensen AM. 2012 Two types of Tet-On transgenic lines for doxycycline-inducible gene expression in zebrafish rod photoreceptors and a gateway-based tet-on toolkit.
PLoS One. 2012;7(12):e51270

Erin said...

Hi I'm Erin Booton from Josh Gamse's lab at Vanderbilt. Thank you for the tol2 kit2.0 distribution. We've gotten many constructs from this kit to transform properly and successfully made preps of them. However 2 in particular are giving me trouble. They're the middle entry clones with loxP-stop-loxP (#727) and #721 which has loxP-mCherry-stop-loxP. Could you (or anyone) tell me if they've had the same problem? I got very small colonies on my Kan plates after transformation then almost no growth or plasmid subsequently. Thanks so much.

Kristen Kwan said...

Hi John,
Thanks for posting! I will spread the word. And of course I don't mind you posting about your reagents!
Best to you and Abbie,
Kristen

Kristen Kwan said...

Dear Erin,

I'm sorry to hear of your troubles. We are happy to send you replacement plasmids. In fact, we found that we mistakenly spotted #721 in the Tol2kit v2.0 - that construct has been updated to a newer version (#759) in which the mCherry cassette has a Kozak sequence to facilitate translation.

Email me (kristen.kwan@genetics.utah.edu) your address information (and FedEx account number if you would like us to use FedEx), and we will send you #727 and #759.

Best,
Kristen

Anonymous said...

This is Greg from the Cornell Lab at the University of Iowa. I have had a few lines I have generated that show proper transient expression in F0. Found F1 by trans-genesis marker. PCR screen of F1 indicates the construct is there, but in-situ does not show the transcript. Any thoughts? Thank you.