Master mix reduces time to results – Thermo Fisher Scientific
The TaqMan Fast Environmental Master Mix Beads give scientists a freeze-dried PCR enzyme formulation for real-time performance.
It can be used with custom or pre-optimized TaqMan Assays on the Applied Biosystems 7500 Fast Real-Time PCR or the StepOnePlus Real-Time PCR.
DNA samples can be added directly to the tubes of pre-aliquotted, ready-to-use reagent and the beads can reduce preparation time and workflow processes by up to 43%, claims the firm.
Once initiated, real-time PCR reactions run in a 40-minute cycle.
Beads come in an eight-well strip-tube format, ready to use individually or in a 96-well footprint.
Lab workflow
Derek Grillo, senior product manager, told FoodQualityNews.com in the Life Technologies lab, they replicated typical workflow and tracked the number of pipetting steps required to make a master mix and incorporate an internal positive control using individual wet reagents.
“It was amazing to see how many steps can be reduced for a lab technician when an all-in-one, lyophilized format is used rather than standard wet reagents,” he said.
“TaqMan Fast Environmental Master Mix Beads not only save labs precious time, but the elimination of pipetting steps also minimizes the risk of contamination and other potential handling errors.”
Formulation and manufacturing steps in the development of the beads are proprietary, said Grillo.
“[However], the same buffer and enzyme formulation used in our widely-sold MicroSeq pathogen detection assays for food and environmental applications are used in TaqMan Fast Environmental Master Mix Beads,” he said.
“This experience coupled with rigorous experiments on common food and environmental sample helped to optimize this master mix for pathogen detection using qPCR.”
It confronts samples that are highly inhibitory to PCR and works across a variety of sample types.
The formulation gives results with different template quantities, down to samples that contain a single copy of a target and highly multiplexed assays.
Grillo added that some of the most common inhibitors in environmental samples are hematin, humic acid, polyphenols, and polysaccharides.
TaqMan Master Mix is manufactured with lyophilisation (freeze-dried) technology which gives researchers a ready-to-use master mix, eliminating messy preparation and error-prone pipetting steps.
IPC and E.coli DNA
The beads are available with and without an internal positive control (IPC) and Grillo said the use of an IPC is an effective way to insure the accuracy of a lab’s results.
“Specifically, an IPC can be used to distinguish true target negatives from PCR inhibition. Although not all labs utilize an IPC, it is becoming more and more necessary as many detection method standards require it.”
The Master Mix Beads may contain low levels of E. coli DNA and when asked about the process if they are used to detect the pathogen, Grillo said recombinant DNA polymerases used in on-market master mix contain residual host genomic DNA (gDNA).
He said although typical purification processes get rid of most residual gDNA, removing 100% is nearly impossible to achieve.
“We have found that manufacturers who claim to have ‘DNA-free’ master mixes may be applying misleading definitions of ‘DNA-free’; we have tested several on-market products and found them to contain easily detectable E.coli gDNA.
“For those labs using TaqMan Fast Environmental Master Mix beads to detect E. coli, we have included in the protocol suggestions for interpretation of amplification data to guide investigators.”
