Biomeme Portable qPCR - General

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 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.

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:

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.

A typical QPCR reaction on the biomeme platform contains the following components