- Before starting
- Assay design
- Preparing primer/probe mix
- Preparing mastermix
- Bulk mastermix
- Lyo GoStrips
- Reaction set-up with Bulk LyoDNA or LyoRNA
- Run set-up
The protocols below detail how we’ve been using Biomeme Inc’s bulk reagents for QPCRs. Once you’ve optimized the assays using bulk reagents, assays can then be manufactured as room-temp stable QPCR assays for use in the field. For the most part, we keep our mastermix and PCR conditions the same across all assays. The variations come with the primer/probe design and optimizing extaction of DNA or RNA from different sample types. So far, we’ve had good success with the primers/probes listed below and with extraction of nucleic acid from nasal and skin swabs.
The only difference between DNA and RNA protocols are the BLB buffer in the extraction kit, and the type of master mix you will use (LyoDNA versus LyoRNA).
The hand-held QPCR device made by Biomeme, called The Franklin Three9, accomodates 9 tubes and three colors/tube: FAM, Cy5/ATTO647N and TexasRed. Cy5 is the brightest channel on the three9, so consider designing the target you anticipate being present in the lowest concentration and/or least sensitive PCR on that channel. FAM is the next brightest, followed by TexasRedX. If you are designing a triplex assay for use with the three9 that includes a positive control, use TexasRedX for the positive control.
For single assays, choose your target gene/region, and use PrimerQuest from IDT to identify suitable forward, reverse and probe sequences. For assays panels involving 2-3 targets, you can still use PrimerQuest to get suitable primer/probe sequences for each of your targets, but you should then use IDT's OligoAnalyzer tool to check for homo- and hetero-dimers for all your primer pairs. Generally avoid any that have a delta G less then -8. If you're trying to multiplex more than 3 targets in a panel, consider using DNAsoftware to design the panel. It's expensive, but you need professional guidance for developing multiplexed assays that are going to work.
Preparing primer/probe mix
20x primer/probe stock contains 4 µM of the forward primer and probe, and 8uM of the reverse primer in TE pH 8.0. Final concentrations in the PCR reaction will be 200 nM of forward and probe, 400 nM of reverse. If starting with 100 µM stocks, then prepare 100 µl as follows:
- 84 uL TE
- 4 uL of forward
- 4 uL of probe
- 8 uL of reverse
Resuspend bulk lyophilized LyoDNA, LyoRNA or LyoGreen master mix with 135 ul of PCR grade water (BEB buffer from the DNA extraction kits is water). This makes a 10x stock. Add 18 ul of glycerol (using a truncated pipette tip) to allow leftover mastermix to be frozen for reuse later. LyoRNA master mix contains a reverse transcriptase for QPCR assays where the target is a RNA molecule. Each resuspended vial contains enough mastermix for at least 60, 20ul reactions.
Lyo GoStrips contain Lyo MasterMix reagent lyophilized in the bottom of PCR strip tubes. No preparation is needed. Simply add DNA or RNA extract, add primers/probes, and start the reaction.
Reaction set-up with Bulk LyoDNA or LyoRNA
A typical QPCR reaction on the biomeme platform contains the following components
10x LyoDNA, LyoRNA or LyoGreen mastermix
isolated DNA or RNA
qs to 20
If you want to use LyoGreen for measuring gene expression, you’ll need to add RT enzyme. We use the Maxima H Minus Reverse Transcriptase from ThermoFisher (200U/ul) at 10U/rxn (0.05ul/20ul rxn)
- To begin, download the Biomeme GO app from either the Apple App store (iPhone) or from the Google Play store (android phone)
- Once installed, open the Biomeme GO app and follow the instructions for using your phone's Bluetooth connection to pair with the Biomeme QPCR machine. If you experience issues connecting the phone, see help documentation here
- To run any assays, you’ll need to either use an existing data folder, or set up a new data folder by selecting the ‘data management’ button on the main screen. Name your folder, create any subfolders if necessary and save.
- Optional: Back on the main app screen, choose ‘protocol management’ to set up a protocol that will contain your thermocycling conditions. If you're just using the standard LyoDNA or LyoRNA, and don't want to store a custom protocol, then you can skip this step.
- You can learn more about how to use Biomeme GO app to manage runs and set-up custom protocols here
Be sure to select a RT step at the start of a protocol if you are testing for RNA targets
- Give your protocol a name and select ‘save’ in the upper right corner of the app
- To begin a run, go back to the main screen of the app and choose ‘start run’. Choose your protocol and data folder, then follow the on screen instructions to begin the run.