Filtered S-cells were allowed to regenerate on MYM medium, from which three regenerated bald colonies (R3, R4 and R5) were selected. After two rounds of growth on MYM, bald colonies of the three strains were grown in TSBS for 2 days at 30 °C, and genomic DNA was isolated from these strains47 . Primers were designed to amplify the infB (BOQ63_RS29885) and atpD (BOQ63_RS19395) genes located in the chromosome, and four genes located on the KVP1 megaplasmid: allC (BOQ63_RS01235), tetR (BOQ63_RS02930), parA (BOQ63_RS04095) and orf1 (BOQ63_RS04285) (Supplementary Table 8 ). The PCR reactions were performed in duplicate in accordance with the manufacturer’s instructions, using 5 ng of DNA, 5% DMSO and the iTaq Universal SYBR Green Supermix Mix (Bio-Rad). Quantitative real-time PCR was performed using a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). To normalize the relative amount of DNA, the wild-type strain was used as a control, using the atpD gene as a reference.
Itaq universal sybr green supermix mix
Manufactured by Bio-Rad
ITaq Universal SYBR Green Supermix is a ready-to-use PCR reaction mix containing all the necessary components for quantitative real-time PCR analysis using SYBR Green detection. The mix includes iTaq DNA polymerase, SYBR Green I dye, dNTPs, MgCl2, and stabilizers.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using itaq universal sybr green supermix mix
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Comparative Genome Analysis of Bald Strains
2
Quantitative Real-Time PCR for Bacterial Genes
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Aliquots of 5 ng of DNA were used as a template in quantitative real time PCR. We used primers for both chromosomal (atpD and infB) and KVP1 (allC, tetR, parA and orf1) genes (Table 2 ). The PCR reactions were performed with the iTaq Universal SYBR Green Supermix Mix (Bio-Rad) using 5% DMSO, according to the manufacturer’s instructions. Reactions was performed in duplicate using a CFX96 Touch Real-Time PCR Detection System (Bio-Rad). To normalise the relative amount of DNA, the wild-type strain was used as a control, using the atpD gene as a reference.
Primers used for quantitative real time PCR
| Primer | Sequence (5′–3′) |
|---|---|
| qPCR_infB-Fw | GTCACGTCGACCACGGTAAG |
| qPCR_infB-Rv | CACCGATGTGCTGGGTGATG |
| qPCR_atpD-Fw | TTCGGACAGCTCGTCCATAC |
| qPCR_atpD-Rv | ACATCGCGCAGAACCACTAC |
| qPCR_parA-Fw | CGGTCGTCACCCAGTACAAG |
| qPCR-parA-Rv | TAACCGAGTTCGAGGGACAG |
| qPCR-orf1-Fw | GAGGGAGCCAATCCCGTATC |
| qPCR-orf1-Rv | GGCTGTTGGACAGGACCATC |
| qPCR-allC-Fw | CGGCGATAGCGGAGACTAAG |
| qPCR-allC-Rv | CCACTGGTGGGACCAGAAAG |
| qPCR-tetR-Fw | TGCTCGACCAGCTGTTGAAG |
| qPCR-tetR-Rv | TGGCGAGCATGAAGTCGTAG |
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