For Info:
Clemencia Colmenares, Ph.D.
Co-Director, Research Core Services
(216) 445-6613
colmenc@ccf.org

CWRU Direct Contacts:
Patrick Leahy, Ph.D.
Core Facility Manager
Phone: (216) 368-0761
Email: pxl24@case.edu

Dr. Martina Veigl
Scientific Director
Phone: (216) 844-7525
Email: mlv2@case.edu

http://www.geacf.net/

Services

Affymetrix

The Affymetrix GeneChip system monitors the expression levels of a large number of genes in a single experiment. It is used to scan the genome for any genes which may be up- or down- regulated in a biological system of interest. Samples of RNA from experimental and control conditions are reverse transcribed into double stranded cDNA, which is then used to synthesize biotinylated cRNA. The Core will assist with cRNA preparation for an additional fee. The Affymetrix technology is provided by the GEACF Core at Case, now located at the Wolstein building. Visit the GEACF web site at http://www.geacf.net/.

Real-time PCR

The ABI 7700 Sequence Detection is a real-time, quantitative PCR system. It monitors the PCR reaction at each cycle of the run and estimates the log phase cycles. It compares the curve generated to that of a known standard and calculates a starting copy number. The system consists of a 96-well thermal cycler with a built in fluorometer. The software packages for quantitation and primer design are installed on a G3 Macintosh.

Users are responsible for purchasing their own kits and consumables and the core maintains the system and offers directions on use and protocols. There are a large number of protocols that employ Taqman chemistries. These include DNA quantitation and RT-PCR using a dual-labeled sequence-specific probe, which is cleaved by the exonuclease activity of the TAQ polymerase and detected by the fluorometer. Other methods employ SybrGreen, which intercalates with double stranded DNA and is detected as the product is generated. There is also a protocol for allelic detection, which is used for identifying known mutations or polymorphisms. Assays for a number of genes have been developed, along with a new Assays-by-Design program available from Applied Biosystems.

Using the system: In order to maximize the use of the machine during the day, we have restricted the times to allow at least 5 runs per day: at 9:00 am, 11:30 am, 2:00 pm, 4:30 pm, and 7:00 pm. Each run will take approximately 2 hours.

To schedule TAQMAN in Outlook:

  1. Go to File/New/Appointment.
  2. Select Invite Attendees from the Actions menu and type "LRI_Taqman" in the To field.
  3. Type your name and phone number in the Subject field.
  4. Type your 12 digit activity number in the Location field.
  5. To look for open time, click on the Scheduling tab.
  6. Once the time has been selected, click Send.
  7. If accepted, you will receive an email confirming the date and time of the appointment.

To reserve time on the machine, use the Outlook "schedule" function. Clicking on "schedule appointment" will allow you to send an appointment to "taqman". To check for available times, click on the "busy?" icon on the lower right. In the dialog box that comes up, be sure to change the length of the appointment to 30 minutes (or 0.5 hours), and the latest time (day should be from 8 am to 8 pm). Notice that all other times will appear busy. You will sign up for only 30 minutes, as the rest of the time has been automatically reserved. Please be sure to include your name, telephone number and activity number in the message. A notice will be returned to you confirming the appointment.

Each run on the instrument is $15.00. If you have a 7pm appointment and think you might be late, please contact Earl Poptic to arrange for the room to remain open.