Self Service Real Time PCR
Equipment
- Two Life Technologies QuantStudio 12K Flex instruments are available for use by appointment. Both instruments accommodate:
- Standard 96 well (200 ul) plates
- 384 well plates
- Taqman Low Density Arrays (TLDA) microfluidic cards
- We do NOT have the 96 well (100 ul) “fast” block.
- The 12K Flex machines can run in fast mode using the standard plate with fast master mix, reducing your run time, if your assay has been optimized for fast mode.
- This equipment may be used for SNP genotyping, gene expression, CNV analysis and microRNA assays.
Location
- Blalock 1003 (Desktop) & Blalock 1005 (Laptop)
- **During COVID-19 Dedensification, please knock on Blalock 1005 and someone will let you into the room. Please maintain 6 feet separation during this process.
Reservations
- Scheduled on a first come, first served, basis using the iLab scheduling module.
- Detailed instructions for using iLab are available. You must be a registered user and attached to your PI’s account in order to use the scheduling module. Everything your PI or financial administrator needs to know about setting up a lab in iLab is on the Core-in-a-box site.
- **COVID-19 Dedensification Restrictions:
- The desktop instrument is available from 6 AM to 4 PM for trained users (runs ending at 6 PM), 7 AM to 3 PM for new users (runs ending by 5 PM)
- The laptop instrument is available from noon to 4 PM.
- There will be a $40 fee applied to all customers who extend past their reservation time, including the time necessary to retrieve data. You must be done by the time your reservation ends.
- There is an automatic 1 hour buffer between reservations. You will not be charged for this buffer.
- Investigators may reserve the machines by the hour, for assays recording real-time metrics, or in 15 minute time increments for end-point reads.
Usage Instructions
- All reactions should be set up by the investigators in their own labs and the plate brought at the appropriate step in the process for loading on the machine.
- The specialized centrifuge and staking equipment necessary for loading the microfluidic cards is available for use in our lab.
- At the completion of the run, investigators should return and retrieve their data on a flash drive.
- A typical gene expression run requires a 2.5-hour reservation to ensure that you have enough time to fill out run details and retrieve data.
- A fast run typically requires a 1.5 hour reservation
- SOPs for inexperienced users are available.
Consumables:
- Consumables for the runs may be ordered through the Core Store.
- MicroAmp® Optical Adhesive Film (catalog 4311971) or the equivalent
- MicroAmp® Optical 384-Well Reaction Plate (catalog 4343370 or 4309849) or the equivalent
- Applied Biosystems Array Card (custom or off the shelf)
- MicroAmp® Optical 96- Well Reaction Plate (N8010560) or the equivalent
- Applied Biosystems TaqMan Reagents or the equivalent
- Applied Biosystems SYBR Green Reagents or the equivalent
- Taqman Gene Expression, MicroRNA, Genotyping and CNV assays may be ordered through our Life Technologies ordering tool.
- Generic assay primers may be ordered through any of our oligonucleotide ordering portals.
Notes for users:
- Please arrive on time for your time slot. It is best to arrive about 5 minutes early. If you start your run late and it runs past the time you have reserved, you will be charged $40 plus the additional time.
- Use only optical plates and lids that are designed for this instrument. Use of other plates and covers may adversely affect your data and our equipment.
- Please do not write with marker on the top of the plate. Write ONLY on the sides of the plates, otherwise the marker will contaminate the machine.
- If you reserve a time slot and don’t call to cancel it at least an hour prior to use, you will be charged for the time slot.
- If you are using the 6 AM slot, you can set up your reactions the previous evening, store them wrapped in foil at 4 degrees overnight and bring them in the morning.
- Data should be analyzed using Expression Suite from Life Technologies. There is also a cloud based version of this software that can handle larger sample sizes.
- Our instruments are typically calibrated to quantitate FAM, VIC & SYBR (or their analogs). The instruments are capable of visualizing up to three other dyes, however, so if you have a specialized experiment, please contact us to discuss it.
- If you are new to qPCR and would like to learn more about it, ThermoFisher has a great YouTube Channel with answers to many questions.
Pricing:
- $20 per hour (internal JHU customers)
- $10 for a 15 minute end point read
- $40 overtime usage fee in addition to actual time used (this includes time spent collecting data). You must be done in the lab by the end of your reserved time.