[ccp4bb] ssDNA self-aneal
Dear all, Recently,i purchase Oligonucleotide from company.the sequence is TTGCGTAC GCAC GTACGC .i want to perform self-annealing process to form the following second structure . 5' TTGCGTACGC ||| |||] 3' CGCATGCA i hope that most of the Oligonucleotide can form this structure,how can i achieve this? any adivice is appreciated. best wishes.
Re: [ccp4bb] ssDNA self-aneal
Just like you would for PCR: - Dissolve the oligonucleotide in water - Heat it to above meltung temperature, pick 95degree C if you want to be on the save side - leave it to cool down. Is this what you are looking for? Tim On Thu, Mar 17, 2011 at 07:23:07PM +0800, dengzq1987 wrote: Dear all, Recently,i purchase Oligonucleotide from company.the sequence is TTGCGTAC GCAC GTACGC .i want to perform self-annealing process to form the following second structure . 5' TTGCGTACGC ||| |||] 3' CGCATGCA i hope that most of the Oligonucleotide can form this structure,how can i achieve this? any adivice is appreciated. best wishes. -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen phone: +49 (0)551 39 22149 GPG Key ID = A46BEE1A signature.asc Description: Digital signature
[ccp4bb] US Distributed Computing Summer School (fully funded)
[Forwarded on behalf of organizers. You may need to be US-based to participate. They are specifically interested in contacting life sciences researchers.] Hello! We invite you and your colleagues to apply now for the 2011 OSG Summer School where you can learn to harness the power of distributed computing while spending a week at the beautiful University of Wisconsin-Madison. During the school, you will learn to use high-throughput computing (HTC) systems--at your own campus or using the national Open Science Grid (OSG)--to run large-scale computing applications that are at the heart of today’s cutting-edge science. Through lectures, discussions, and lots of hands-on activities with experienced OSG staff, you will learn how HTC systems work, how to run and manage lots of jobs and huge datasets to implement a scientific computing workflow, and where to turn for more information and help. The school is ideal for graduate students in computer science or other sciences where large-scale computing is a vital part of the research process, but any qualified and interested applicant will be considered. In the past, we've had students from diverse backgrounds including genetics, geographic information systems, and physics. Successful applicants will have all travel and school expenses paid for by the OSG. Furthermore, as part of a collaboration with TeraGrid, students will go to the annual TeraGrid Conference (TG11, July 18–21, 2011 in Salt Lake City, Utah) with all travel and conference expenses paid. Important dates: Applications Open: Now Applications Close: Friday, April 1, 2011 School Session: June 26-30, 2011 For more information, please visit http://www.opensciencegrid.org/GridSchool or email us at gridschool-2011-i...@opensciencegrid.org. We hope to hear from you soon. Sincerely, Tim Cartwright and Alain Roy 2011 OSG Summer School Organizers
Re: [ccp4bb] ssDNA self-aneal
I would bring up the DNA in TM buffer (10 mM Tris, 5 mM MgCl2) or similar and anneal under dilute conditions to favor hairpin formation over dsDNA. Fast cooling will also favor hairpin formation, so you may try heating to 95° and then cooling on ice, or using a short gradient on a thermocycler. You can check your work on native PAGE (including appropriate controls, like oligos annealed under dsDNA-favoring conditions) On Thu, Mar 17, 2011 at 4:23 AM, dengzq1987 dengzq1...@gmail.com wrote: Dear all, Recently,i purchase Oligonucleotide from company.the sequence is TTGCGTAC GCAC GTACGC .i want to perform self-annealing process to form the following second structure . 5' TTGCGTACGC ||| |||] 3' CGCATGCA i hope that most of the Oligonucleotide can form this structure,how can i achieve this? any adivice is appreciated. best wishes.
Re: [ccp4bb] ssDNA self-aneal
Has anyone looked at the kinetics of DNA annealing. Especially for such short fragments I expect hairpin formation times to be on the order of pico to nano seconds. Of course it doesn't hurt to slow-cool but I wouldn't be too paranoid about it. Moreover, in this particular case the hairpin is probably sufficiently unstable to release and refold at a high rate at room temp so even less need to slow cool. A bigger concern may be the relative affinity of the intra-molecular hairpin versus the inter-molecular "primer dimer". The former benefits from lower entropy loss, but the latter would not need such a tight loop and possibly would form one additional G-C basepair. Bart On 11-03-17 12:06 PM, Kevin Jude wrote: I would bring up the DNA in TM buffer (10 mM Tris, 5 mM MgCl2) or similar and anneal under dilute conditions to favor hairpin formation over dsDNA. Fast cooling will also favor hairpin formation, so you may try heating to 95 and then cooling on ice, or using a short gradient on a thermocycler. You can check your work on native PAGE (including appropriate controls, like oligos annealed under dsDNA-favoring conditions) On Thu, Mar 17, 2011 at 4:23 AM, dengzq1987 dengzq1...@gmail.com wrote: Dear all, Recently,i purchase Oligonucleotide from company.the sequence is TTGCGTACGCACGTACGC .i want to perform self-annealing process to form thefollowing secondstructure . 5'TTGCGTACGC ||| ||| ] 3'CGCATGCA i hope that most of the Oligonucleotide can form this structure,how can i achieve this? any adivice is appreciated. best wishes. -- Bart Hazes (Associate Professor) Dept. of Medical Microbiology Immunology University of Alberta 1-15 Medical Sciences Building Edmonton, Alberta Canada, T6G 2H7 phone: 1-780-492-0042 fax:1-780-492-7521
[ccp4bb] (i)mosflm indexing / cell refinement
I seem to have noticed that (i)mosflm can index on one image, or two images that are not related by 90 degrees. also, cell refinement sometimes splits up a set of frames into maybe 3 or four segments of e.g. a few degrees each. and of course, I can set it based on frames 90 degrees apart. reason I ask is that i thought mosflm's modus operandi was based on frames in general being related by 90 degrees - is mosflm simply written differently or is there some criteria it uses to make these settings, or something else... -Bryan
Re: [ccp4bb] (i)mosflm indexing / cell refinement
afaik Mosflm will index on whatever spots you have told it to find and not discard, be they from 1 image, 2, or 10 or more images at any relative angle or oscillation range. Or it won't successfully index, depending on the difficulty of the case (several lattices, spot overlap etc...). Indexing from two images at 90 degrees from each other is simply an easy good practice rule that often, but not absolutely always, works. Many times a single image is more than enough, sometimes many images and a lot of trying is necessary to get the right indexing matrix. Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3, Campus Cantoblanco E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/content/research/macromolecular/mvraaij/index.php?l=1 On 17 Mar 2011, at 19:44, Bryan Lepore wrote: I seem to have noticed that (i)mosflm can index on one image, or two images that are not related by 90 degrees. also, cell refinement sometimes splits up a set of frames into maybe 3 or four segments of e.g. a few degrees each. and of course, I can set it based on frames 90 degrees apart. reason I ask is that i thought mosflm's modus operandi was based on frames in general being related by 90 degrees - is mosflm simply written differently or is there some criteria it uses to make these settings, or something else... -Bryan
[ccp4bb] X-ray kit seeks good home
We have a very low mileage rotating anode (Rigaku RU-H3RHB 18KW Cu anode, 0.3mm focus) which has been retired for a while now following arrival of an Xcalibur. There are also lots of spares, some new. And a table top enclosure. Free to anyone who is able to come and get it. Trevor Greenhough -- Professor Trevor J. Greenhough Director of PG Research, ISTM Faculty of Health RT Coordinator ISTM and School of Life Sciences Huxley Building Keele University Keele ST5 5BG Tel 01782 733405 / 734331 (Lisa) email t.j.greenho...@keele.ac.uk
Re: [ccp4bb] determining the domain for overexpression and crystallization
Dear all, Thank you very much for all of information about domain determination! I think the limited proteolysis is a good choice for the domain determination as we have no information about a protein. However, if we do have a domain information by the bioinformatics, how can we truncate the domain? Are we need to keep three or five more amino acids in two ends? Thanks. Best regards, Xianhui On Tue, Mar 8, 2011 at 2:22 AM, Mark J van Raaij mjvanra...@cnb.csic.eswrote: for an experimental way to determine soluble domains see the following paper: ESPRIT: an automated, library-based method for mapping and soluble expression of protein domains from challenging targets. Yumerefendi H, Tarendeau F, Mas PJ, Hart DJ. J Struct Biol. 2010 Oct;172(1):66-74. Epub 2010 Mar 4. Review. PMID: 20206698 [PubMed - indexed for MEDLINE] Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3, Campus Cantoblanco E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/content/research/macromolecular/mvraaij/index.php?l=1 On 7 Mar 2011, at 19:18, gauri misra wrote: Hi, To start with it would be great if you look in to the secondary structure prediction of the sequence using any of the standard servers like PSIPRED, JPRED etc. Many more available at expasy site http://ca.expasy.org/tools/. Whatever construct you finally choose to make just remember the standard rule that we generally follow is to avoid deleting the alpha helices and beta sheets. You can design your initial primers so as to obtain the complete amplification of these secondary structures from any part within the protein. You can even use the various modules of the following online available server http://scratch.proteomics.ics.uci.edu/ to have an idea of the intrinsically disordered regions in the protein, transmemebrane regions and disulfide bonds that would certainly help you in initiating in the right direction. Best wishes Gauri On Mon, Mar 7, 2011 at 4:10 AM, Xianhui Wu wuxian...@gmail.com wrote: Dear all, Before we try to study the crystal structure of an unknown protein, we need to determine the sequence that can fold into a compact and stable 3D domain. What kinds of methods can we choose? -- Best regards, XH Wu -- Best regards, Xianhui
[ccp4bb] postdoc position in Singapore
A postdoctoral position is available at the Laboratory of Virus Structure and Function in the Duke-NUS Graduate Medical School, Singapore. The Laboratory of Shee-Mei Lok (http://www.duke-nus.edu.sg/web/research_faculty_sheemei.htm) is looking for a post-doctoral fellow to work on the structural studies of dengue viruses and its complexes. Her laboratory uses a combination of techniques such as x-ray crystallography and cryo-electron microscopy (CryoEM) including single particle analysis and cryotomography. Facilities such as in-house x-ray machines and cryoelectron microscope (Titan Krios, FEI) are available in the National University of Singapore. The post-doctoral fellow will mainly participate in the computational side of structure solving and should preferentially have prior experience in solving either cryoEM or x-ray structures. Training in crystallography or cryoEM data collection and image processing however, will also be available if necessary. Interested individuals are invited to send cover letter, cv and a list of referees to Dr Shee-Mei Lok by email (sheemei@duke-nus.edu.sg).
Re: [ccp4bb] determining the domain for overexpression and crystallization
yes limited proteolysis is the best choice for the domain determination of unknown protein from our lab some people did this. after doing limited proteolysis just sequence digested part N terminally or the C terminally and find out the region from where its getting digested. side by side u can do fold prediction using phyre or u can use fold index to know the compact part of the protein. go through J Biol Chem. http://www.ncbi.nlm.nih.gov/pubmed/18974091 2008 Dec 26;283(52):36532-41. Epub 2008 Oct 30. Characterization of Rv3868, an essential hypothetical protein of the ESX-1 secretion system in Mycobacterium tuberculosis. Luthra Ahttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Luthra%20A%22%5BAuthor%5D , Mahmood Ahttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Mahmood%20A%22%5BAuthor%5D , Arora Ahttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Arora%20A%22%5BAuthor%5D , Ramachandran Rhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Ramachandran%20R%22%5BAuthor%5D . did similar work . On Fri, Mar 18, 2011 at 4:19 AM, Xianhui Wu wuxian...@gmail.com wrote: Dear all, Thank you very much for all of information about domain determination! I think the limited proteolysis is a good choice for the domain determination as we have no information about a protein. However, if we do have a domain information by the bioinformatics, how can we truncate the domain? Are we need to keep three or five more amino acids in two ends? Thanks. Best regards, Xianhui On Tue, Mar 8, 2011 at 2:22 AM, Mark J van Raaij mjvanra...@cnb.csic.eswrote: for an experimental way to determine soluble domains see the following paper: ESPRIT: an automated, library-based method for mapping and soluble expression of protein domains from challenging targets. Yumerefendi H, Tarendeau F, Mas PJ, Hart DJ. J Struct Biol. 2010 Oct;172(1):66-74. Epub 2010 Mar 4. Review. PMID: 20206698 [PubMed - indexed for MEDLINE] Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3, Campus Cantoblanco E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/content/research/macromolecular/mvraaij/index.php?l=1 On 7 Mar 2011, at 19:18, gauri misra wrote: Hi, To start with it would be great if you look in to the secondary structure prediction of the sequence using any of the standard servers like PSIPRED, JPRED etc. Many more available at expasy site http://ca.expasy.org/tools/. Whatever construct you finally choose to make just remember the standard rule that we generally follow is to avoid deleting the alpha helices and beta sheets. You can design your initial primers so as to obtain the complete amplification of these secondary structures from any part within the protein. You can even use the various modules of the following online available server http://scratch.proteomics.ics.uci.edu/ to have an idea of the intrinsically disordered regions in the protein, transmemebrane regions and disulfide bonds that would certainly help you in initiating in the right direction. Best wishes Gauri On Mon, Mar 7, 2011 at 4:10 AM, Xianhui Wu wuxian...@gmail.com wrote: Dear all, Before we try to study the crystal structure of an unknown protein, we need to determine the sequence that can fold into a compact and stable 3D domain. What kinds of methods can we choose? -- Best regards, XH Wu -- Best regards, Xianhui -- Vandana Kukshal Postdoc Fellow structure biology group international Center For Genetic Engineering and Biotechnolgy Aruna Asaf Ali Marg 110 067 New Delhi INDIA
