tag:blogger.com,1999:blog-7158647951423191205.post1543367276577291083..comments2023-09-29T05:46:05.022-06:00Comments on Tol2kit: Questions on the Tol2kit, 4/26/08-6/8/09Chi-Bin Chienhttp://www.blogger.com/profile/04543802635501556885noreply@blogger.comBlogger78125tag:blogger.com,1999:blog-7158647951423191205.post-64549433010477595452015-07-27T13:12:01.088-06:002015-07-27T13:12:01.088-06:00Hi,
This Pr. M'hamed Grati,
I am starting lear...Hi,<br />This Pr. M'hamed Grati,<br />I am starting learning about zebrafish transgenesis. I have a construct that I generated where I put the GFP under the control of a zebrafish cell type specific promoter/enhancer. I injected the linearized version of it into fish embryos, and obtained very sporadic cell type specific expression of the reporter, which is good news. Now, I want to border this construction with Tol2 5' and 3' sites. I wonder if you could provide me the primers (or full sequence) that I should use to amplify these fragments on zebrafish genomic DNA. Besides, I wonder if you could tell me where to obtain transposase mRNA for co-injection to zebrafish embryos. Thank you very much in advance for the help!<br />Please email me your feedback to m.grati@med.miami.edu since I rarely look at my Google account.M'hamed Gratihttps://www.blogger.com/profile/13518883345508823957noreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-45903005779482400122009-11-21T05:58:50.520-07:002009-11-21T05:58:50.520-07:00This comment has been removed by a blog administrator.Anonymousnoreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-45234501820806646282009-06-09T11:43:36.003-06:002009-06-09T11:43:36.003-06:00Hi,
Thanks for the help getting VNTI to recognize...Hi, <br />Thanks for the help getting VNTI to recognize pDest vectors (see 4/8/09)! FYI- Pasting the sequences works fine, but the attR3 and attR4 sites have to be annotated, and in the right orientation :).<br />-MollyMolly Nyholmnoreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-6555318604457858672009-06-08T21:39:09.118-06:002009-06-08T21:39:09.118-06:00Clara-
There are some minor fluctuations in the at...Clara-<br />There are some minor fluctuations in the att sequences, especially the longer ones like attR and attP. It is not clear whether these are errors in the Invitrogen documentation, or perhaps undocumented improvements from one version to the next of the Gateway kit. I just ignore single bp differences in the long att sites; everything still seems to work. However, if you are concerned, the p5E-MCS sequence is likely to come from direct sequencing and therefore to be correct, while the attR1 sequence comes from documentation and therefore is less certain to correspond to reality..Chi-Bin Chienhttps://www.blogger.com/profile/04543802635501556885noreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-55330253924371682102009-06-08T16:05:51.538-06:002009-06-08T16:05:51.538-06:00Qing--
If your 5' clone is only enhancer, pro...Qing--<br /><br />If your 5' clone is only enhancer, promoter, 5' UTR, and possibly intron (no start codon), the reading frame obviously won't matter; the starting frame will be set by the first start codon (presumably pME-EGFP or something similar).<br /><br />We typically use proofreading Taqs (usually Phusion or the Takara long-amplification polymerase) that do not leave T tails.Chi-Bin Chienhttps://www.blogger.com/profile/04543802635501556885noreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-802887305153015442009-06-08T15:57:06.916-06:002009-06-08T15:57:06.916-06:00Filipe--
You didn't say how big a construct y...Filipe--<br /><br />You didn't say how big a construct you were trying to build. In general I would expect inserts up to 10 kb or so to work pretty well, and then would expect variable results above that. For very large constructs, we have used a construct p5E-FA that has 8-cutter (FseI, AscI) sites in a 5' clone, then clone in conventionally.Chi-Bin Chienhttps://www.blogger.com/profile/04543802635501556885noreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-17591301217856981292009-06-08T15:57:03.664-06:002009-06-08T15:57:03.664-06:00Filipe--
You didn't say how big a construct y...Filipe--<br /><br />You didn't say how big a construct you were trying to build. In general I would expect inserts up to 10 kb or so to work pretty well, and then would expect variable results above that. For very large constructs, we have used a construct p5E-FA that has 8-cutter (FseI, AscI) sites in a 5' clone, then clone in conventionally.Chi-Bin Chienhttps://www.blogger.com/profile/04543802635501556885noreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-75385190918730641642009-06-08T15:47:15.831-06:002009-06-08T15:47:15.831-06:00Hi Molly!
Here's a comment from Jonathan Rose...Hi Molly!<br /><br />Here's a comment from Jonathan Rosen back on 8/11/08:<br /><br />----<br />Jonathan Rosen said...<br />I figured out the problem, which I will explain here in case anyone else has the same issue. Vector NTI wasn't recognizing the ccdB sequence in the vector, so I had to put it in manually by going to Feature Map --> add. After this, Vector NTI treated it as a destination vector.<br />----<br />I think the issue may be that there is a (nondeleterious) point mutation in the ccdB of all the destination vectors, which either came from Invitrogen in pDEST R4-R3 or was introduced at the very beginning of our multisite work. This single bp change prevents Vector NTI from recognizing the ccdB<br />--Chi-BinChi-Bin Chienhttps://www.blogger.com/profile/04543802635501556885noreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-74355496141707018842009-06-08T11:44:47.952-06:002009-06-08T11:44:47.952-06:00Hi Molly,
Instead of pasting the sequence into VN...Hi Molly,<br /><br />Instead of pasting the sequence into VNTI, try saving the annotated Genbank format sequence from the Wiki as text and then importing it. Alternatively, you might just check the file you already have to make sure the attB sites are annotated as "Misc. recombination" features, that might be the problem.<br /><br />Good luck,<br />Jimjim listerhttps://www.blogger.com/profile/13150067785966909254noreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-27891667285983172052009-06-08T10:09:11.954-06:002009-06-08T10:09:11.954-06:00Hi,
Does anyone use vector NTI to make their gatew...Hi,<br />Does anyone use vector NTI to make their gateway plasmid maps? Our lab standardly uses this software, and I would like to draw all of our new clones in this software. <br /><br />I can create new entry clones (perform BP reactions) fine in VNTI, but when I try to perform LR reactions the software doesn't recognize the Destination vecotrs from the tol2 kit as destination vectors, although it does recognize that they have attb3 and attB4 sites. I am creating the VNTI files by pasting pDEST sequences from the Tol2 site. Any hints on how I get VNTI to recognize pDEST vectors?<br /><br />Thanks, <br />MollyMolly Nyholm, Grinblat Labnoreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-86933879190085261892009-06-04T09:36:04.689-06:002009-06-04T09:36:04.689-06:00Hi,
I am trying to make a very large construct an...Hi, <br />I am trying to make a very large construct and I am not being able to.<br /><br />Briefly I do a typical 10um Lr reaction O.N. and then I clean the reaction with a Quiagen reaction cleaning kit(PCR kit), I resusped in 10ul and I use 5ul for transformation.<br /><br /> I am using electrocompetent CopyCutter bacteria from Epicentre for that porpuse but something is going wrong as I do not get any colony. <br /><br />I wonder if anyone has any protocol that could share with me.<br /><br />Thank you very much for your help<br /><br />Filipe SousaFliphttps://www.blogger.com/profile/12816449438210933306noreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-75929817882346108922009-05-26T08:00:07.424-06:002009-05-26T08:00:07.424-06:00Hi, I want to make put my promoter of different le...Hi, I want to make put my promoter of different lengthes in the 5'entry clone and make Promoter-GFP constructs. Do I have to consider the reading frame shift problem? What if I put the promoter-exon1-intron1-part of exon2 in the 5'entry clone?<br /><br />Which is the most commonly used Taq? Will the Taq introduce an additional A into the PCR product and will this influence the multisite recombination and reading frame? <br /><br />Thanks a lot!Qinghttps://www.blogger.com/profile/18200827300594312818noreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-17585150902991616312009-05-12T07:06:00.000-06:002009-05-12T07:06:00.000-06:00There seems to be a lack of congruence between the...There seems to be a lack of congruence between the attR1 sequence and the p5_MCS whole sequence. While attR1 appears as ...ctatgaaTcaactactta..., p5_MCS shows the following sequence ...ctatgaaCcaactacttag...<br /><br />Probably the mistake is in the vector whole sequence, I am right?Claranoreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-11913948388909494382009-04-22T06:29:00.000-06:002009-04-22T06:29:00.000-06:00Hi,
I have been trying to recombine three differ...Hi, <br /><br />I have been trying to recombine three different PCR products in pDONRP4-P1r. After the BP reaction and transfo of bacteries that are not tolerant to ccdb, we have thousands of colonies, but none of them contains the insert... since we are sure the vector we use contains the cassette, it seems that we have a intramolecular recombination (this phenomen occurs even when we do not pu any insert in it, just enzyme)... Did it already happen to you? what can be the solution to promote the insertion of my product?<br /><br />thanks a lot, <br /><br />Emilie.Emilie M.noreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-82563260111883542042009-04-14T14:05:00.000-06:002009-04-14T14:05:00.000-06:00Hi,
I received the pDONRP4-P1R vector with the ...Hi,<br /> I received the pDONRP4-P1R vector with the kit. When I transformed the vector on ccdB tolerant cells, I obtained two kinds of colonies on the plate. A predominant population of small translucent colonies and a minor number of opaque and large colonies. I first thought that the ccdB competent cells were contaminated. I transformed the other donor vectors and I got uniform kind of colonies. I performed the self complementation screen of Lawson lab on the P4-P1R vector. I picked up 8 colonies and none of them seem to be the right ones. Could you please help me?<br />Thank you<br />Best wishes<br />Arularulhttps://www.blogger.com/profile/07583957637764562930noreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-83070203765707846052009-03-23T12:21:00.000-06:002009-03-23T12:21:00.000-06:00Has anyone generated a p3E-IRES-H2A-EGFPpA that th...Has anyone generated a p3E-IRES-H2A-EGFPpA that they wouldn't mind sharing. Thanks, Chris Sansam (sansamcl@mit.edu)Chris S.https://www.blogger.com/profile/17238603079748969580noreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-19479621145906011182009-03-22T15:03:00.000-06:002009-03-22T15:03:00.000-06:00Brian-That would be useful, but we don't have such...Brian-<BR/>That would be useful, but we don't have such clones. I can probably find a clone containing destabilized GFP for you, if you'd like to make a middle clone yourself.Chi-Bin Chienhttps://www.blogger.com/profile/04543802635501556885noreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-71310123949858998532009-03-19T17:10:00.000-06:002009-03-19T17:10:00.000-06:00Has anybody made entry clones containing destabili...Has anybody made entry clones containing destabilized GFP or RFP/mCherry? We are looking to test the activity of promoters driving Gal4, so ideally we'd like UAS:destabilized Fluor construct.Anonymousnoreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-37441838729712146272009-03-04T10:36:00.000-07:002009-03-04T10:36:00.000-07:00Geoff--Sorry, I wasn't at the meeting and so did n...Geoff--<BR/><BR/>Sorry, I wasn't at the meeting and so did not hear the talk. The other potential advantage, besides possibly improved integration rates, is that you should presumably get clean single-copy BAC integrations, which would be easy to map by inverse or LM-PCR.Chi-Bin Chienhttps://www.blogger.com/profile/04543802635501556885noreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-11699240448714061942009-03-03T14:58:00.000-07:002009-03-03T14:58:00.000-07:00Chi Bin,Thanks for the information. Do you recall ...Chi Bin,<BR/><BR/>Thanks for the information. Do you recall what percentage of potential founders sent the BAC germ-line?<BR/><BR/>Cheers, <BR/>GeoffAnonymousnoreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-78180694125453538712009-03-03T14:45:00.000-07:002009-03-03T14:45:00.000-07:00Geoff--Indeed, Koichi Kawakami has placed Tol2 end...Geoff--<BR/><BR/>Indeed, Koichi Kawakami has placed Tol2 ends into a BAC using bacterial homologous recombination and used this construct for transgenesis. I believe that he presented this work at the recent Asilomar PI meeting.Chi-Bin Chienhttps://www.blogger.com/profile/04543802635501556885noreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-33124244515290782832009-03-03T14:15:00.000-07:002009-03-03T14:15:00.000-07:00Zhiqiang--All of the current Tol2kit clones are li...Zhiqiang--<BR/><BR/>All of the current Tol2kit clones are listed on the wiki. We have thought about including IRES-RFP clones in future releases. IRES-mCherry is likely to be quite dim.<BR/><BR/>Chi-BinChi-Bin Chienhttps://www.blogger.com/profile/04543802635501556885noreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-10293541668656163362009-03-03T12:15:00.000-07:002009-03-03T12:15:00.000-07:00Hi, We are trying to make a handful of different B...Hi, <BR/><BR/>We are trying to make a handful of different BAC transgenics and wanted to learn if anyone had tried putting ISceI or Tol2 sites around a BAC insert and then tested for increased germ-line transmission frequency. <BR/><BR/>Thanks, <BR/>Geoff Burns<BR/>MGH, Boston, MAAnonymousnoreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-37249945475413456652009-03-03T11:17:00.000-07:002009-03-03T11:17:00.000-07:00Hello, Prof Chien, does the most recent vision of ...Hello, Prof Chien, does the most recent vision of Tol2 kit include the 3' entry vector p3E-IRES-RFP(or dsRed)? <BR/><BR/>Does any one have this plasmid, and is willing to gift some to us?<BR/><BR/>Zhiqiang Zeng, Edinburgh Cancer Research Centre (zq.zeng@ed.ac.uk)greenfishhttps://www.blogger.com/profile/14922382900901689415noreply@blogger.comtag:blogger.com,1999:blog-7158647951423191205.post-57908504318594298572009-03-03T02:10:00.000-07:002009-03-03T02:10:00.000-07:00Sorry, my full information is Jon Hildahl at the A...Sorry, my full information is Jon Hildahl at the Alestrom zebrafish lab/Norwegian School of Veterinary Science, Ullevålsveien 72, PO Box 8146 Dep, 0033 Oslo, Norway. I'll check around to see if I can find a new clone, but if you know of anyone in the area I'd be greatly interested. Thanks again for the help with this excellent blog. <BR/><BR/>/JonAnonymousnoreply@blogger.com