Very Important Consumable Requirements
The instrument requires very specific consumables, plates, tips, oil, Supermix. Not using the required supplies can damage the instrumentation severely.
Absolutely no SYBR! It will destroy the droplet reader!
CGRB supplies the required plastics and oil which is recovered in the fees. Supermix is included in the full service fee structure. For multi-user service you may purchase the supermix yourself. Consult with staff to make sure you have the correct reagents ahead of time.
After scheduling your run with CGRB staff and submitting an order to Web Order, you may pick up your consumables to run your samples. Check in with staff before the run to let them know the number of samples you will be running and to start access to the instrument.
Reserve time to use instrument. Current schedule is available here. Time requested will show up after approved.
You must receive training by CGRB staff prior to operating this instrument independently. Please contact staff to schedule training.
Service maintained by Anne-Marie Girard and Katie Carter.
Example Sample Setup
Here is an example of setup of samples, NTC, positive control setup, to be run in triplicate. ddPCR – Sample Preparation Outline.
- Samples should be arranged in columns and in an Eppendorf twin.tec semi-skirted 96 well plate. (example: catalog #951020362). Other plates will not work with the ADG. Plates are available from CGRB online store on an individual basis if needed.
- Set up of ddPCR reactions similar to qPCR in initial experimental setup parameters.
- Cut the DNA to make it easier for the enzyme to find templates. You can try reactions without cutting first, but cutting often will increase “yield”. Is also recommended for regular qPCR. Use NEB website to search for sites that don’t cut your region of interest.
- Run should include: No Template Control, Negative Control, Positive control (aim for 100,000 copies of Target) in triplicate.
- Set up positive control spatially far from NTC and Negative control in the plate.
- Run dilutions of target to determine optimal concentration. Run triplicates (to control for user pipetting error and instrumentation variability.) Dilutions get rid of inhibitors and can make ddPCR more sensitive than qPCR. Optimal target concentration is 1-5 copies/droplet. May be able to get rid of triplicates once best dilution determined for assay.
- Make as big a master mix as possible. Avoid pipetting 1 µl. Never make individual reactions.
- Very important to thoroughly mix master mix. Vortex master mix and vortex after samples added. Thermal mixing in tube will not distribute master mix as in regular PCR.
- Droplet reader uses negative control to do calculation, set up threshold
- You must use Bio-Rad reagents! Do not use SYBR green! Its fluorescence will destroy the droplet reader and your PI will be responsible for replacement. Use EvaGreen instead.
- You may set up your reactions in your own lab and walk them over here.
- Strongly suggest Rainin tips or some other high quality tip. Low quality tips have sheared ends which can have small fragments that interfere with the droplet generation.
- Recommended amplicon length is 60-200 bp. BioRad has gotten 1.5KB EvaGreen and 4.4KB Taqman probes to work in-house. Out in the field have seen 2500KB EvaGreen and 5000KB Probes.
- Use FAM and HEX, or FAM and VIC. Probe is hydrolyzed during hybridization
- ddPCR™ Supermix for Probes (No dUTP) gives stronger reactions that the version with dUTP.
- Optimization of reaction: Annealing temperature could be changed +/- 10°C from the 60°C. Also can change primer and probes. Make serial dilutions to find the optimal concentration of template.
- SYBR based chemistry (EvaGreen) with primers constructed 100-200 bases apart may possibly be optimized to see different populations in the results.
- Assay design: Default concentration for primer 900nm to drive reaction to completion.