From c.sinensis from gmail.com Thu Aug 7 07:04:46 2008 From: c.sinensis from gmail.com (Zhang Yi) Date: Thu Aug 7 09:38:31 2008 Subject: [Drosophila] FRT-directed deletions PCR genotyping? Message-ID: <200808072004448592224@gmail.com> 2008-08-07 Do anybody have experience on making FRT-directed deletions using Exelixis stocks described in Park et al 2004 Nat.Genetics paper? I did one WH minus - RB plus deletion, but failed to find any correct deletion in 96 lines via PCR. My PCR was using similar primers described in Park et al paper. I did 20ul rxn with Takara LA Taq. Annealing temperature touchdown from 68c, 65c, 62c, 59c (each 2 cycle) then 56c 25 cycles. I used 1 min annealing and 72c, 12min extension. The product should be ~7.3kb according to the paper, but I failed to get any band in between primer and the well. However there were thick bands in the well. Could anybody help me to figure out what is the problem? Thanks a lot! Best, Yi -- No fate but what we make. Zhang Yi Lab of Neuroscience, B109 National Institute of Biological Sciences, 7 Kexueyuan Rd. ZGC Life Sciences Park, Beijing 102206 China, People's Republic of http://algebra.yculblog.com From Liam.Keegan from hgu.mrc.ac.uk Wed Aug 13 08:07:07 2008 From: Liam.Keegan from hgu.mrc.ac.uk (Liam Keegan) Date: Wed Aug 13 11:33:29 2008 Subject: [Drosophila] Three year PhD Studentship in MRC Human Genetics Unit, Edinburgh Message-ID: <48A2DC7B.3090602@hgu.mrc.ac.uk> Dept/School Human Genetics Unit, Medical Research Council Project Title Drosophila Screen for Regulators of RNA editing Project Supervisor(s) Dr L Keegan, Dr M O’Connell Application Deadline 22 August 2008 The MRC Human Genetics Unit is offering a three year PhD studentship funded by the Scottish Motor Neurone Disease Association working under the supervision of Dr Liam Keegan. This excellent opportunity would suit a student finishing an MSc project on Drosophila or a student graduating with a good training in Genetics and Developmental Biology and some experience of working with Drosophila or another efficient model organism. The project is based in lab studying RNA editing. This is a step in RNA processing that affects the expression levels and the amino acid sequences of vertebrate glutamate receptors. Loss of RNA editing has been reported in motor neurones of ALS patients (Kawahara (2004) Nature 427, 801). and following ischaemia in central neurons (Peng (2006) Neuron 49, 719). Glutamate is the major excitatory neurotransmitter in vertebrate CNS and loss of the edited forms of vertebrate glutamate receptors is expected to promote neuron death by allowing excessive calcium influx. Vertebrates have multiple genes encoding the relevant RNA editing enzymes, called ADARs, whereas the fruit fly Drosophila has a single Adar gene. The project will take advantage of Drosophila to carry out a genetic screen for proteins that interact with and regulate the activity of these RNA editing enzymes. As in vertebrates flies mutant for the Adar gene lose RNA editing in a range of target transcripts encoding ion channel subunits. The flies have locomotion defects from birth, reduced viability and show age-dependent neurodegeneration. All phenotypes are rescued by expression of either Drosophila or human ADARs. Adenosine to inosine RNA editing targets stretches of double stranded RNA which may be either short hairpins within pre-mRNAs or long stretches of dsRNA formed by transposable elements or by antisense pairing of complementary transcripts. Site-specific editing within pre-mRNAs affects RNA splicing and recodes open reading frames to produce ion channel variants. Non-specific editing within long dsRNAs antagonizes the RNA interference that is triggered by these dsRNAs. The screen will potentially contribute to understanding both these roles of ADARs. The screen will identify regulators of ADAR activity in the fly by looking for modifiers of viability in Adar loss of function mutants that have reduced viability already or by looking for suppressors of lethality caused by early overexpression of ADAR (Keegan (2005) Embo J 24, 2183). The screen uses a standard, efficient method in Drosophila that begins with looking for effects of larger heterozygous deficiencies and then refining the effects down to specific genes. The objective is to identify regulators that are conserved and likely to be informative about the regulation of editing in vertebrate neurons and to focus on the roles of these in regulation of ADAR activity and in neuroprotection. Kawahara (2004) Nature 427, 801 Peng (2006) Neuron 49, 719 Keegan (2005) Embo J 24, 2183 Lab information is available at: www.hgu.mrc.ac.uk/users/Liam.Keegan/Home.html www.hgu.mrc.ac.uk/Research/OConnel/crrt.html Funding Notes This 3 year PhD studentship will be funded by the Scottish Motor Neurone Disease Association and we ask potential applicants to note that while there are eligibility criteria for this studentship, we encourage all qualified EU students to apply. Further information regarding eligibility can be found on www.hgu.mrc.ac.uk/StaffInf/Studentships/residency.html The studentship offers an attractive stipend and a comprehensive transferable skills training programme. Applicants should submit a covering letter explaining why you are interested in applying. Applications should include a full up-to-date C.V. (include vacation address), and names and addresses of two academic referees, sent by 22 August 2008 to: student-admin@hgu.mrc.ac.uk or Studentships, MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh EH4 2XU. -- Dr. Liam Keegan Tel No. ++ 44 (0)131 467 8417 MRC Human Genetics Unit Fax No. ++ 44 (0)131 467 4856 Western General Hospital Crewe Road Edinburgh EH4 2XU UK http://www.hgu.mrc.ac.uk/ http://www.hgu.mrc.ac.uk/users/Liam.Keegan/Home.html http://www.hgu.mrc.ac.uk/Research/OConnel/ From heterochromatic from gmail.com Thu Aug 21 13:35:42 2008 From: heterochromatic from gmail.com (sandra schulze) Date: Thu Aug 21 13:41:46 2008 Subject: [Drosophila] needle pullers Message-ID: <5b26f17e0808211135q40b359fbw5bc6f7f4f8a56117@mail.gmail.com> Hello Flyland, Well it's time to buy a needle puller. I have only ever used the Sutter horizontal P-97 with two stage pulling, and that machine costs about $9000 all told. I am very cautious about buying a cheaper machine having never had any experience with any other make, but am balking at the $9000 price tag of the Sutter. Please send me your feedback about needle pullers you have known and loved. The machines, I mean. Many thanks, Sandra From hrundle from uottawa.ca Mon Aug 25 13:14:47 2008 From: hrundle from uottawa.ca (hrundle@uottawa.ca) Date: Mon Aug 25 13:31:59 2008 Subject: [Drosophila] dispensing food Message-ID: <68c097cd-ea4d-4d14-a5ad-ed3ba2389b36@b1g2000hsg.googlegroups.com> Hi, We've cook fly media in small batches and have been dispensing it using a Mettler-Toledo 323DZ peristaltic pump. However, some new food recipes we are using in the lab (by necessity - we can't switch) are too thick for this pump and do not dispense consistently. A "Droso- filler" (i.e. fills 100 vials at once) does not make sense because we cook in such small volumes (1.5L/cook). We therefore need to either scale up our cooking operation somewhat, or find a better pump. I would love to hear any recommendations or advice on either: 1. setting up a medium scale cooking operation. What type of pot/ kettle would you recommend? We are the only fly lab in the department, so we're looking at cooks on the scale of 4-10L I would think. We don't have easy access to a steam supply either. 2. getting a better pump that deals well with thick food. Any information would be much appreciated! Howard Rundle Department of Biology University of Ottawa Ottawa, ON Canada From scottatg from aol.com Tue Aug 26 12:10:17 2008 From: scottatg from aol.com (scottatg@aol.com) Date: Tue Aug 26 13:45:01 2008 Subject: [Drosophila] Re: Dros Digest, Vol 40, Issue 4 In-Reply-To: <200808261704.m7QH45U03868@net.bio.net> References: <200808261704.m7QH45U03868@net.bio.net> Message-ID: <8CAD5B5BF775644-1660-37F7@webmail-db07.sysops.aol.com> watson marlow 520 series pumps will work the best. www.watsonmarlow.com ?Ask me about Miele Glassware Washers. Best Regards, Scott Saveleski Atlantic Technology Group, Inc. 1906 North Hamilton Street, Suite G Richmond, Va. 23230 1-800-359-5153 x2 410-371-8740 cell -----Original Message----- >From: dros-request@oat.bio.indiana.edu >To: dros@magpie.bio.indiana.edu Sent: Tue, 26 Aug 2008 1:04 pm Subject: Dros Digest, Vol 40, Issue 4 Send Dros mailing list submissions to dros@net.bio.net To subscribe or unsubscribe via the World Wide Web, visit http://www.bio.net/biomail/listinfo/dros or, via email, send a message with subject or body 'help' to dros-request@net.bio.net You can reach the person managing the list at dros-owner@net.bio.net When replying, please edit your Subject line so it is more specific than "Re: Contents of Dros digest..." Today's Topics: 1. dispensing food (hrundle@uottawa.ca) ---------------------------------------------------------------------- Message: 1 Date: Mon, 25 Aug 2008 11:14:47 -0700 (PDT) >From: hrundle@uottawa.ca Subject: [Drosophila] dispensing food >To: bionet-drosophila@net.bio.net Cc: hrundle@uottawa.ca Message-ID: <68c097cd-ea4d-4d14-a5ad-ed3ba2389b36@b1g2000hsg.googlegroups.com> Content-Type: text/plain; charset=ISO-8859-1 Hi, We've cook fly media in small batches and have been dispensing it using a Mettler-Toledo 323DZ peristaltic pump. However, some new food recipes we are using in the lab (by necessity - we can't switch) are too thick for this pump and do not dispense consistently. A "Droso- filler" (i.e. fills 100 vials at once) does not make sense because we cook in such small volumes (1.5L/cook). We therefore need to either scale up our cooking operation somewhat, or find a better pump. I would love to hear any recommendations or advice on either: 1. setting up a medium scale cooking operation. What type of pot/ kettle would you recommend? We are the only fly lab in the department, so we're looking at cooks on the scale of 4-10L I would think. We don't have easy access to a steam supply either. 2. getting a better pump that deals well with thick food. Any information would be much appreciated! Howard Rundle Department of Biology University of Ottawa Ottawa, ON Canada ------------------------------ From atc12 from mole.bio.cam.ac.uk Wed Aug 27 09:27:57 2008 From: atc12 from mole.bio.cam.ac.uk (Adelaide) Date: Wed Aug 27 09:36:57 2008 Subject: [Drosophila] Re: dispensing food References: Message-ID: <48B5646C.CFA5DD40@mole.bio.cam.ac.uk> For small batches, I find using *any* pump to be more trouble than it is worth: I pour them by hand, both the thick and the thin. I use a one liter plastic beaker with a sharp lip, never try to pour from more than ca 300 ml at a time, and arrange the setup so that I never have to pour across more than 4 vials in a tray (my trays hold 9 rows, so I pour the right-side 5 rows then turn the tray around to pour the remaining 4). With a little practice one gets pretty good at uniformity but it's never perfect, so I set the too-little ones aside and use them for storing virgins and fertility tests. Takes me 5 minutes per 100 vials: and no pump to clean up afterwards! And I cook all my fly food over a gas hob -- pan just needs to be large enough so that when the food boils up it doesn't boil over, nothing special there. Hope this helps, and feel free to contact me directly with any further questions! Cheers, Adelaide Carpenter hrundle@uottawa.ca wrote Mon, 25 Aug 2008 11:14:47 -0700 (PDT) > > Hi, > > We've cook fly media in small batches and have been dispensing it > using a Mettler-Toledo 323DZ peristaltic pump. However, some new food > recipes we are using in the lab (by necessity - we can't switch) are > too thick for this pump and do not dispense consistently. A "Droso- > filler" (i.e. fills 100 vials at once) does not make sense because we > cook in such small volumes (1.5L/cook). We therefore need to either > scale up our cooking operation somewhat, or find a better pump. > > I would love to hear any recommendations or advice on either: > > 1. setting up a medium scale cooking operation. What type of pot/ > kettle would you recommend? We are the only fly lab in the department, > so we're looking at cooks on the scale of 4-10L I would think. We > don't have easy access to a steam supply either. > > 2. getting a better pump that deals well with thick food. > > Any information would be much appreciated! > > Howard Rundle > Department of Biology > University of Ottawa > Ottawa, ON > Canada From kcook from bio.indiana.edu Thu Aug 28 11:43:19 2008 From: kcook from bio.indiana.edu (Kevin Cook) Date: Thu Aug 28 13:00:08 2008 Subject: [Drosophila] Postdoc at the MRC Centre for Neurodegeneration Research Message-ID: <200808281645.m7SGjISp012927@mail-relay.iu.edu> 3 year Postdoctoral position available at the MRC Centre for Neurodegeneration Research, London, UK Applications are invited for the post of Postdoctoral Research Worker to work on a project based at the MRC Centre for Neurodegeneration Research, Institute of Psychiatry, King's College London. The mission of the MRC Centre is to further understand the mechanisms of neurodegeneration and to find new treatments for diseases such as Alzheimer's, Parkinson's and motor neurone disease. An experienced and motivated scientist is required to investigate the role of TDP-43 dysfunction in a Drosophila model of motor neurone disease. The project will include generation and maintenance of mutant and transgenic flies, DNA and RNA purification, northern, southern, and western blotting, protein biochemistry, in situ hybridization and immunocytochemistry, loss-and gain-of-function mutant analyses as well as characterisation of behavioural phenotypes of genetically modified Drosophila. Practical expertise with Drosophila cultures as well as broad genetic, biochemical and molecular biology laboratory experience are essential. Candidates will be expected to hold a PhD or equivalent. This three year MRC-funded position is available from November 2008 until October 2011. Starting salary will be ?31,461 pa (inclusive of London Allowance, and pending pay award), depending on qualifications and experience. To obtain further particulars and further information about the Institute, please see our website at http://www.iop.kcl.ac.uk/vacancies or alternatively email vacancies@iop.kcl.ac.uk. Applications, in the form of a CV (including details of two referees), covering letter and equal opportunities statement, should be emailed to vacancies@iop.kcl.ac.uk. Please quote reference number 08/R86 in all correspondence. Closing date for applications 15 September 2008. Only candidates shortlisted for interview will be contacted. Equality of opportunity is College policy