The MCIC provides services for

• Capillary sequencing using the ABI 3100xl
• Genotyping: fragment analysis on Beckman CEQ8800, SNP analysis on Luminex200 or Illumina BeadXpress
• Deep genome sequencing using the Illumina Genome Analyzer II.

Molecular biology equipment available at the MCIC

Real-time quantitative PCR

Instrumentation. A Bio-Rad iQ5 is available to users for real-time PCR experiments. It enables to monitor PCR product accumulation on line during the PCR reaction in presence of a fluorescent dye (SYBR Green) or of fluorescently labeled oligonucleotide probes (Hybridization probes). The Bio-Rad iQ5 uses 96-well reaction format and up to five targets can be multiplexes in one well. For more information on the iQ5 capabilities and consumables visit: http://www.bio-rad.com under Life Science Research, and http://www.biocompare.com/gonl.asp?id=114482 for multiplexing kits.

Procedure for using the iQ5

Instructions and tip for running real-time PCR on the iQ5

• Our instrument is calibrated for Bio-Rad microtiter plates and optical tape (Bio-Rad, PCR Plastic Consumables, Cat#2239441, with optical tape Cat# 2239444), therefore you need to use these plates and tape. If not, the instrument would need to be re-calibrated.
• Use PCR or Rt-PCR kits that contain fluorescein as internal standard for well factor correction. Fluorescent dye Rox containing kit are now available on the market, but his dye can not be read by our instrument. You can use a Rox dye containing kits, which does not interfere with data collection, but you can not perform dynamic well factor correction, and you will need to set up an external well factor plate or use persistent well factors for data normalization.
• When you cover your plate with the optical tape, make sure that you do not touch the tape with your hands, or gloves: grease from your hands or powder from the gloves may deposit on the optical film and cause wrong fluorescence readings. To completely seal the plate, press the optical tape down using the protective paper strip that you peeled off the tape, or use a roller (there is one in the drawer near
  the iQ5 machine).
• Do not keep your plates in direct contact with ice, because ice or droplets of water may be transferred into the iQ5 block. This will cause wrong fluorescence readings (think of lens effect). Your plate has to be completely dry before you put it in the iQ5 machine. Deposit the plate into the iQ5 block gently, and don’t press it down into the block wells.
• If you do not have a full 96-well microtiter plate, you can cut it, and run strips. You have two choices: (1) if the strip does not have the side skirt, place it in the middle of the block so that the heated lid presses it down straightly, or (2) place it at one end of the block filling the empty spaces with the empty strips available to you near the machine. You need to do this second option, if your strip has the side skirt.
• In the plate set up window, choose the reaction volume of 20 ul, because the wellfactor correction module is for a 20 ul reaction. You can still run a slightly different volume, for example 15 ul or 25 ul, but make sure that samples that will be compared have the same volume.
• In the Run set-up window, choose the dynamic mode for well-factor corrections, by selecting “Collect well factor from experimental plate”, if you are using a kit that contain fluorescein as internal well standard.
• For data analysis, chose the “PCR baseline subtraction” as Analysis Mode.
• During the run, do not use other programs on the iQ5 computer as this may interfere with the data collection.

Some references

For a tutorial on realtime PCR: http://pathmicro.med.sc.edu/pcr/realtime-home.htm
For a tutorial on cDNA synthesis go to: http://www.dur.ac.uk/biological.sciences/Staff/Croy/cDNAfigs.htm
A good reference for beginners is: Fraga, D., Meulia, T. and Fenster, S. Real-time PCR. 2007. Chapter 32 in ‘Current Protocols in Essential Laboratory Techniques.’ John Wiley & Sons Publishing.

If you are ordering Rio-Rad supplies for your PCR work, use the Bio-Rad quotation 07-Q29825V2 to receive the University discout. The quote lists the discounted item, and it can be used by anyone on the OARDC campus using the Bio-Rad OARDC account number. Download Bio-Rad 07-Q29825V2 quote.

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QPix

Instrumentation. The Genetix QPix is a benchtop colony picking and gridding/macroarraying robotics used for both high throughput mapping and sequencing, and also for preparation of quality genomic or cDNA libraries for microarraying or other screening. Capabilities:

QPix picking function automatically picks colonies or plaques from culture trays (petri dishes, 22cm2 bioassay trays or omnitrays) and then it inoculates them into 96 or 384 well plates. The QPix can pick up to 3,500 colonies per hour. A CCD camera is used to image the colonies or plaques. The user then specifies the picking threshold related to the color, roundness and proximity of the clones and a digital script is written. The computer then guides the robot's picking arm, equipped with a 96 pin picking head, in the colony picking process. Once the 96 pins have picked colonies, the arm inoculates pre-filled 96 or 384 well microtiter plates. The picking head is then sent to the washing station for washing and sterilization, and then it is ready for the next picking round.

QPix gridding function transfers and arrays colony libraries from culture plates onto filter membranes. The gridding source are 96 or 384 well microtiter plates and the destination filters can be put down on wetted filter blocks or onto agar trays.

Similarly, the QPix can replicate libraries by transferring colonies from source 96 or 384 microtiter culture plates onto 96 or 384 destination plates.

If you would like to use the QPix please first contact the mcic staff.

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PhosphorImager

Instrumentation. The Molecular Dynamics Storm840 imaging system is capable of producing digital images of blots labeled with radioisotopes or chemiluminescence (blue-excitable chromophors).
To detect radioactive samples, the blot is first exposed to a phosphor screen which records the image, which is then read by the scanner. The exposure time is approximately 1/10 of that of a autoradiogram film, and the linear range is 1 to 100,000. Film has a range of 1 to 500.
The Storm840 also detects fluorescence of dyes that are excited at 450nm and have emission specra longer than 520nm. It detects chemiluminescence of samples labeled with alkaline phosphatase and the substrate AttoPhos(TM).
Find more information about this system at http://www.mdyn.com, under "products" and then "Storm840".

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ArrayWoRx Biochip Reader

Instrumentation. The Applied Precision ArrayWoRX scanner uses white light and a CCD camera to image nucleic acid or protein arrays on glass slides. Capabilities:

• The instrument can image simultaneously up to 4 colors
• Wavelength Flexibility (350nm - 750nm)
• Standard Filter Sets Include Cy3, Cy5, Alexa 488 (FITC), Alexa 350 (UV)
• Supports Industry-Standard Slide:
  - Corning CMT GAPS II
  - Erie Sciences Gold Seal
  - Amersham Codelink
• User-Defined Resolution Sampling from 3.25um to 26um
• The SoftWoRX software included with the scanner allows you to manage your experiments form beginning to end as well as analyze your images and export data to third party data analysis programs.

For additional information visit the Applied Precision web site: http://www.api.com/lifescience/arrayworx.html. Check out also the FAQs on this site (http://www.api.com/lifescience/arrayworx_FAQs.html), which are very informative.

Biomek3000

This instrument is a versatile liquid handling robot that performs repetitive tasks with high accuracy and reproducibility of the manipulations in less time. Capabilities

Methods for performing the following tasks are available for Biomek-3000 users:

• DNA extraction from plant leaves using the Wizard¨ Magnetic 96 DNA Plant System (Promega cat#FF3761). Contact MCIC staff to get discount rate for the kit.
• Pooling PCR products from several plates.
• Setting PCR reactions, including adding DNA and Master Mixes.

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