Western University BiologyWestern Science

Molecular Genetics Unit (MGU)

Students working in MGU

Two students examining a DNA print out in MGU

MGU core provides a number of facilities in the field of plant and animal molecular biology, molecular genetics and general life science research. The following faculty members use the facility on daily basis: Dr. Shiva Singh (Molecular Genetics of Complex disease and phenotypes), Dr. Kathleen Hill (Mutation analysis, genome organization and Integrity), Dr. Greg Kelly (Cell signaling in Vertebrate embryo), Dr. T Percival-Smith (Molecular Mechanisms of Morphogenesis, Drosophila melanogaster ), and Dr. V Grbic (Arabidopsis Development Genetics) and Dr. Sashko Damjanovski (Extracellular Matrix remodeling in developing Xenopus laevis ). The MGU Core facility is available for similar research by other Biology Faculty for a user fee. It is located on the third floor of the Western Science Centre (242m2).

It is a large and well-equipped common area (242m2) which includes State-of-art gel documentation facility (alpha Flour Chem 8900 digital imaging of DNA/RNA and Chemi-doc for Western blotting), Real time PCR (Corbett Rotor Gene 2000), Spectrophotometer (Beckman DU 640 for DNA/RNA quantification), Liquid Scintillation Counter (Beckman 3801 and 5801), Animal and Plant Culture rooms, Microscopy and Imaging (Leica DMR BE fluorescence), Ultrapure water system, Steam Autoclaves for sterilization of biological samples, Hot air sterilization ovens, X-ray film processor, Microtome for cry sectioning (Leitz 1720), high speed ultra-centrifuges, cold room, incubators, vacuum pump and concentrators, homogenizers, and laminar flow hoods.

Instruments

Alpha Innotech FlourChem IS-8900

The equipment can be used for a wide array of quantitative imaging applications ranging from chemi-luminescence imaging, fluorescence imaging, gel, film and membrane imaging to culture and microplate-based assays.

The equipment has an excellent image quality combined with an easy to use software features. It has a CCD camera with 1.92 Megapixel Resolution with a 12-bit Dynamic Range which is ideal for chemiluminescence, fluorescence and absorbance imaging. The analysis software can perform a broad range of analysis likes Molecular Weight calculation, Rf determination, 1-D lane densitometry, 2-D spot, quantitative PCR, gel scoring and automated colony counting.

Corbett Research RG 3000

The Corbett Research RG 3000 is a reliable thermal cycler, allowing researchers to perform PCR reactions quickly while also giving them the ability to monitor the reaction in real-time. The cycler holds either 36-0.2 ml or 72-0.1 ml standard PCR tubes and it is an open chemistry platform. The open chemistry platform uses multiple excitation sources (470, 530, 585 and 625 nm LED high power diodes) combined with several detection filters (510, 555 and 610 nm bandpass and 665, 570 and 610 nm high-pass) to detect virtually every known fluorophore (Sybr-Green I, FAM, TET, JOE, VIC, HEX, ROX, TAMRA, CY3, CY3.5, CY5, CY5.5, Oregon Green, CAL Red, Red 640 and Texas Red). Also, most DNA amplification enzymes/ buffers can be used. The system comes with data analysis software.  The main advantage of the RG3000 is the flexibility and the low running cost of the cycler.  The reaction vessels are standard PCR tubes and no expensive kits have to be used.

Beckman DU 640 for DNA/RNA quantification

Beckman DU 640 UV-VIS Spectrophotometer Real nice, microprocessor controlled instrument intended for use in quantitative and qualitative biological research and industrial procedures that require spectrophotometric measurements in the UV visible region of the electromagnetic spectrum -, wavelength range 190-1100 nm. Photometric readout (-0.300)-3.000 A, 0.0-200.0 %T. Scan speed 120, 240, 600, 1200, 2400 nm/min. Standard routine measurements of fixed wavelength, wavelength scan in absorbance and transmittance, and kinetics time absorbance- optional application modes available are protein analysis, nucleic acid, fraction read/plot, single component quantitative analysis, enzyme mechanism, enzyme activity, multi-component analysis, gel scan, and performance validation. 

Incubator Shaker

The New Brunswick Scientific Innova 4300 Large Capacity Incubator Shaker is designed for handling greater quantities of flasks. It easily holds (12) 2 L flasks and accepts flasks up to 6 L.  The Innova 4300 shaker incorporates a triple eccentric counterbalanced drive to provide horizontal plane rotary motion in a 1 in. (25.4 mm) circular orbit. A Proportional/Integral (PI) Microprocessor controller with instantaneous digital feedback controls the speed over a range of 25-500 RPM. It also provides temperature control over a range of 5°C above ambient to 60°C. The internal chamber is 34¾ inches (88.3 cm) wide, 22¼ inches (56.6 cm) deep, 19¼ inches (48.9 cm) above the platform and will accept flasks up to 6 liters.

Sorvall RC-5B Refrigerated Centrifuge

The Sorvall RC-5B, a refrigerated and high speed floor model centrifuge in the family of Sorvall (a division of DuPont), is built to deliver easy operation, exceptional performance, and reliability for tough clinical applications.  The DuPont Sorvall RC-5B centrifuge features a rotor, angular velocity and time/min knob, automatic programmed acceleration, a simple on/off switch, and braking functions. The drive systems of this machine are outfitted with a universal/drive motor. This centrifuge offers quiet operation and has the capability to operate at a speed of 20,000 maximum rpm (48,300x g).

Other Instruments

Animal and Plant Culture rooms, Microscopy and Imaging (Leica DMR BE fluorescence), Ultrapure water system, Steam Autoclaves for sterilization of biological samples, Hot air sterilization ovens, X-ray film processor, Microtome for cry sectioning (Leitz 1720), cold room, incubators, vacuum pump and concentrators, homogenizers, and laminar flow hoods.

User Fee

New users need to sign yearly contract and participate in yearly costs to maintain facility (approximately on average $700-800/year).  The costs are intended to offset anticipated repairs and service contracts, to maintain the lab and equipment in good order.

Policy

Access

MGU is located in Western Science Center WSC, room 357 and it is open on weekdays from 9:00 am until 5:00 pm. During these hours, the Facility manager will be available for training and consultation regarding the instruments. The staff or student card can be registered in order to gain card access to the facility after hours. During after hours, the user will be held responsible for ensuring that doors are kept locked at all times.

Responsibilities of Users and their Supervisors

Users must be trained by the Facility staff to ensure safe usage of the equipment. Log books are provided for each instrument and users are expected to log in their time of usage. When the work is complete, the user should ensure that the work area is left clean. Any problems or concerns can be noted in the log books or the user can contact the Facility manager.  In the event of instrument damage due to abuse, misuse, or unauthorized use, the research supervisor will be held financially responsible for the cost of repair or replacement.

Billing

The facility will charge fees in order to cover the cost of consumables and instrument operating costs as defined in the fee section. Users must provide a valid speed-code or account number for invoicing.

Publications

Nieuwesteeg, M. Walsh, L.A., Fox, M.A. and Damjanovski, S. 2012. Domain specific overexpression of TIMP-2 and TIMP-3 reveals MMP-independent functions of TIMPs during X laevis development. Biochem Cell Biol. r2011-0088.

Walsh, L.A., Cepeda, M.A. and Damjanovski, S. 2012. Analysis of MMP-dependent and independent functions of tissue inhibitor of metalloproteinase-2 on the invasiveness of breast cancer cells. J. Cell Comm.Signal. 2012 Jan 8.

Rosloski SM, Jali SS, Balasubramanian S, Weigel D, Grbic V. (2010) Genomic rearrangements and natural variation in splicing of MADS AFFECTING FLOWERING 2 leads to diverse flowering responses in Arabidopsis thaliana. Genetics 186, 263-276.

Wang Q, Sajja U, Rosloski S, Humphrey T, Kim MC, Bomblies K, Weigel D, Grbic V. (2007) HUA2 Caused Natural Variation in Shoot Morphology of A. thaliana. Curr Biol. 17, 1513-1519.

Poduska B, Humphrey T, Redweik A and Grbic V (2003) The synergistic activation of FLOWERING LOCUS C by FRIGIDA and a new flowering gene AERIAL ROSETTE 1 underlies a novel morphology in Arabidopsis. Genetics 163, 1457-1465.

Smith CA, Newson TA, Leonard KC, Barfett J, Holdsworth DW, Hutnik CM, Hill KA. A framework for modeling ocular drug transport and flow through the eye using micro-CT. Phys Med Biol. 2012 Oct 7;57(19):6295-307.

Ward TL, Prtenjaca A, Hill KA, A novel E. coli-derived mutation detected with the Big Blue ÒcII mutant selectable assay. Environ Mol Mutagen 2010 Jan 29.

Van Osch FS, Piliguian M, Hill KA, Spontaneous mutation frequency is elevated in skin of harlequin (hq)/Big Blue Ò mice. Mutagenesis 2010 Jan 20.

Butler JL, Osborne Locke ME, Hill KA, Daley M. HD-CNV: hotspot detector for copy number variants.  Bioinformatics. 2013 Jan 15;29(2):262-3.

Wen, J.W.H., Hwang, J.T.K., and G.M. Kelly. Reactive oxygen species and Wnt signalling crosstalk patterns mouse extraembryonic endoderm. 2012 Cell. Signal. 24(12):2337-2348. 

Hwang, J.T.K. and G.M. Kelly. GATA6 and FOXA2 regulate Wnt6 expression during extraembryonic endoderm formation. 2012 Stem Cells Dev. 21(17):3220-3232.

Sun, Q. and G.M. Kelly. Post-translational modification of CASK leads to its proteasome-dependent degradation. 2010 Int. J. Biochem. Cell Biol. 42(1):90-97.

Tian, G ., Lu, Q., Zhang, L., Kohalmi, S.E., Cui, Y. 2011. Detection of protein interactions in plant using a gateway compatible bimolecular fluorescence complementation (BiFC) system. Journal of Visualized Experiments doi: 10.3791/3473.

Bross, C.D., Corea, O.R.A., Kaldis, A., Menassa, R., Bernards, M.A., Kohalmi, S.E. 2011. Complementation of the pha2 yeast mutant suggests functional differences for arogenate dehydratases from Arabidopsis thaliana. Plant Physiology and Biochemistry, 49, 882-890.

T. Fatima, C. Snyder, WR. Schroeder, D. Wishart, RJ Weselake and P Krishna. 2012. Transcriptome and metabolite analysis of polyunsaturated fatty acid-enriched sea buckthorn (Hippophae rhamnoides L.) seed. PLoS ONE. 7(4):e34099 (1-18 pages)

Isidro J, Knox R, Singh AK, Clarke F, Krishna P, DePauw R, Clarke J, Somers D. 2012. Brassinosteroid leaf unrolling QTL mapping in durum wheat. Planta Feb 18.

Udaka H, Percival-Smith A, Sinclair BJ.  Increased abundance of Frost mRNA during recovery from cold stress is not essential for cold tolerance in adult Drosophila melanogaster.  Insect Mol Biol. 2013 Jul 31. doi: 10.1111/imb.12044.

Moazzen H, Rosenfeld R, Percival-Smith A. Non-requirement of a regulatory subunit of Protein Phosphatase 2A, PP2A-B', for activation of Sex comb reduced activity in Drosophila melanogaster. Mech Dev. 2009 Aug-Sep;126(8-9):605-10.

Kleiber ML, Mantha K, Stringer RL, Singh SM. Neurodevelopmental alcohol exposure elicits long-term changes to gene expression that alter distinct molecular pathways dependent on timing of exposure. J Neurodev Disord. 2013 Mar 13;5(1):6.

Laufer BI, Mantha K, Kleiber ML, Diehl EJ, Addison SM, Singh SM. Long-lasting alterations to DNA methylation and ncRNAs could underlie the effects of fetal alcohol exposure in mice. Dis Model Mech. 2013 Jul-Aug;6(4):977-92.

Contact

The MGU core facility is managed by Dr. Gurpreet Dhami (gdhami2@uwo.ca), WSC 343, x 86406.