DNA was extracted from the eggshell using a PowerLyzer® PowerSoil® DNA Isolation Kit (Qiagen, Venlo, The Netherlands) according to the manufacturer’s guidelines. The abundance of total bacteria, Firmicutes and Bacteroidetes phyla, Enterobacteriaceae family and Lactobacillus spp. were quantified using the primers and PCR protocols described in Table 1 . Amplification and detection were performed using the CFX384 Bio-Rad Real-time PCR detection system (Bio-Rad, Nazareth, Belgium). Each reaction was done in duplicate in a 12-μl total reaction mixture using 2 x SensiMix SYBR No-ROX mix (Bioline, Luckenwalde, Germany) and 2 μl of DNA.
Sensimix sybr no rox mix
Manufactured by Meridian Bioscience
Sourced in Germany, Belgium
SensiMix SYBR No-ROX mix is a ready-to-use 2X master mix for real-time PCR amplification that utilizes SYBR Green I dye for detection. The mix contains all necessary components for efficient and reliable amplification, including a high-performance DNA polymerase, dNTPs, and stabilizers.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using sensimix sybr no rox mix
1
Bacterial Community Profiling from Eggshells
2
Quantitative Real-Time PCR Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
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The lung and intestinal samples were stored in RNAlater (Ambion, Ghent, Belgium) and RNA was extracted using the Aurum kit (Bio-Rad, Nazareth-Eke, Belgium). cDNA was synthesized using the iScript cDNA synthesis Kit (Bio-Rad) and real time qPCR was performed on the CFX384 detection system (Bio-Rad), using the SensiMix SYBR No-ROX mix (Bioline, Kampenhout, Belgium). The expression levels were normalized to the most stable housekeeping genes, which were determined for each organ using the geNorm Housekeeping Gene Selection Software within qBASE+ (Biogazelle, Zwijnaarde, Belgium) [41 (link)]. The primer sequences are listed in Table 5 .
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