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For comparison, three different immunization schemes were designed: high dose protocol (HDP; 0.5 27 mg of venom), low dose protocol (LDP; 0.1 7 mg of venom) and Mix protocol (MP; preimmunization 0.1 1.2 mg of crotalic PLA2, and then 4.5 8 mg of venom).
Real-time PCR was used to quantify cDNA using an ABI7500 sequence detector and Taqman PCR Master Mix Protocol (ABI, Foster City, CA).
For quantitative PCR, samples were analyzed in 25-µl reactions (10 ng of cDNA, 900 nmol of primer, 12.5 µl of master-mix, and water) according to the standard protocol provided in SyBR® Green PCR Master Mix protocol (Applied Biosystems).
Targets that produce ΔΔCt values within ± 1.5 were considered uniformly preamplified (TaqMan PreAmp Master Mix Protocol, Applied Biosystems).
Roche DIG RNA Labeling Mix protocol was used and staining was visualized using anti-FITC Fab fragments coupled to alkaline phosphatase (1 2000, Roche).
Complementary DNA (cDNA) was produced from 1 μg RNA of each sample using the 'High Capacity RNA to cDNA' master mix protocol (PN 4375575, Applied Biosystem, Foster City, CA, USA).
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For this protocol, Assays A and C (25 µL each) were mixed and treated exactly as in Mixing protocol #1.
Figure 1 shows the mixing protocol.
The mechanical properties were not significantly affected by the organoclay type or the mixing protocol.
Interfacial properties are also dependent on the mixing protocol used to produce the blends.
MWCNT/FKM and GNR/FKM nanocomposites were prepared via a solution mixing/melt mixing protocol.
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