Mastermix Case Study
2.4 Intervention products
During both the run in period and the intervention period, the participants received four slices of bread (100 gram) and two servings of RETC (33.4 gram) each day. The WGW products have a whole grain content of 98.4 gram in total each day. For the weekends small buns, current buns and current bread were provided to the participants. The WGW and RW have a similar energy and macronutrient composition, although the fibre composition is different. The nutritional facts of the bread and the ready to eat cereals (RTEC) are depicted in Appendix 2.4. These intervention products should replace their habitual intake of wheat products. In order to have a well-defined intervention and control, the participants were not allowed …show more content…
A standard qPCR was performed by using a SensiMix real-time PCR reagent (Bioline, Londen, UK), which was used to make a Mastermix. The SensiMix contains all necessary components for the reaction such as enzymes, stabilizers, nucleotides and SYBR green. Besides the SensiMix, the Mastermix also contained a forward and reverse primer which serve as starting point for DNA synthesis. The used primers were designed using PrimerBank, a public resource for PCR primers, by first searching for a NCBI Gene ID and filling in this Gene ID number (http://pga.mgh.harvard.edu/primerbank/). Requirements of appropriate primers and the sequences of the used primers can be found in Appendix 2.6. The Mastermix was added to all 20x diluted samples and one standard curve in a 384 wells plate. Thereafter, a Bio-Rad CFX384 C1000 Thermal Cycler machine (Bio-Rad Laboratories BV, Veenendaal, the Netherlands) was used to run the samples. The thermal program was as follows: 10 minutes at 95°C, followed by 40 cycles of 10 seconds at 95°C + 15 seconds at 60°C, 10 seconds at 95°C followed by melt curve 65°C to 95°C and the plate read. The generated melt curve is used to check if the reaction resulted in specific PCR
During both the run in period and the intervention period, the participants received four slices of bread (100 gram) and two servings of RETC (33.4 gram) each day. The WGW products have a whole grain content of 98.4 gram in total each day. For the weekends small buns, current buns and current bread were provided to the participants. The WGW and RW have a similar energy and macronutrient composition, although the fibre composition is different. The nutritional facts of the bread and the ready to eat cereals (RTEC) are depicted in Appendix 2.4. These intervention products should replace their habitual intake of wheat products. In order to have a well-defined intervention and control, the participants were not allowed …show more content…
A standard qPCR was performed by using a SensiMix real-time PCR reagent (Bioline, Londen, UK), which was used to make a Mastermix. The SensiMix contains all necessary components for the reaction such as enzymes, stabilizers, nucleotides and SYBR green. Besides the SensiMix, the Mastermix also contained a forward and reverse primer which serve as starting point for DNA synthesis. The used primers were designed using PrimerBank, a public resource for PCR primers, by first searching for a NCBI Gene ID and filling in this Gene ID number (http://pga.mgh.harvard.edu/primerbank/). Requirements of appropriate primers and the sequences of the used primers can be found in Appendix 2.6. The Mastermix was added to all 20x diluted samples and one standard curve in a 384 wells plate. Thereafter, a Bio-Rad CFX384 C1000 Thermal Cycler machine (Bio-Rad Laboratories BV, Veenendaal, the Netherlands) was used to run the samples. The thermal program was as follows: 10 minutes at 95°C, followed by 40 cycles of 10 seconds at 95°C + 15 seconds at 60°C, 10 seconds at 95°C followed by melt curve 65°C to 95°C and the plate read. The generated melt curve is used to check if the reaction resulted in specific PCR