The PCR reactions were performed in a final volume of 25 µL, using 12.5 µL of HotStarTaq Master Mix 2X (Qiagen), containing 2.5 units of HotStarTaq DNA polymerase, 1.5 mM of MgCl2 and 200 µL of each dNTP. Then, 1 µM of each oligonucleotide primer and 1 µL of DNA were added. The amplification profile involved an initial denaturation step at 95°C for 5 min, followed by 30 amplification cycles. Each amplification cycle consisted of 30 s at 94°C, 30 s at 51°C and 60 s at 72°C. A primer extension step (72°C for 10 min) followed the final amplification cycle. The positive and negative controls for the extraction were included. The PCR reactions were processed in a Mastercycler Personal (Eppendorf, Hamburg, Germany). All reactions were performed in duplicate.
Hotstartaq master mix 2x
Manufactured by Qiagen
Sourced in Germany
HotStarTaq Master Mix 2X is a ready-to-use mixture of HotStarTaq DNA Polymerase, buffer, and dNTPs optimized for PCR amplification. It is designed for high-specificity and high-sensitivity PCR applications.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using hotstartaq master mix 2x
1
Optimized PCR Amplification Protocol
2
PCR Amplification of DNA Samples
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The PCR reactions were performed in a final volume of 25 µL, using 12.5 µL of HotStarTaq Master Mix 2X (QIAGEN, Hilden, Germany), containing 2.5 units of HotStarTaq DNA Polymerase, 1.5 mM of MgCl2 and 200 µL of each dNTP. Then, 1 µM of each oligonucleotide primer and 1 µL (40 ng/µL) of DNA were added. The amplification profile involved an initial denaturation step at 95°C for 15 min, followed by 30 cycles at 94°C for 30 s, 50°C for 40 s and 72°C for 60 s. The positive and negative controls for the extraction and PCR were included. The PCR reactions were processed in a Mastercycler Personal (Eppendorf, Milan, Italy). All reactions were performed in duplicate.
3
Mycoplasma PCR Identification Protocol
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The extraction of genomic DNA from the broth culture of above field isolates was carried out by boiling, as per (Rauf et al., 2013) . The M. gallisepticum and M. synoviae antigen (Soleil, Biovac, France) were used as positive controls to carry out PCR specific to genus Mycoplasma as well as MG and MS species respectively. The PCR was carried out on extracted DNA samples from field isolates of Mollicutes to amplify the 16S rRNA gene fragment specific to Genus Mycoplasma, as well as M. gallisepticum and M. synoviae species using respective sets of published primers (Zhi et al., 2010; Raviv et al., 2007; OIE, 2008) . Details of the primers used are given in Table 2. The PCR reaction mix (25 µl) contained 0.5 µl of each primer (10 µM), 12.5 µl of Master mix (HotstarTaq master mix 2X, Qiagen), 10.5 µl of nuclease free water (NFW ) and 1 µl of template DNA. Amplification of 16S rRNA specific to Genus Mycoplasma was done using the protocol as described by Zhi et al. (2010) . For amplification of DNA of MG two sets of primers were used. For the first primer (16S rRNA) amplification of M. gallisepticum DNA was done as per the method of (OIE, 2008) and for the second primer (16S-23S rRNA) protocol of (Raviv et al., 2007) was followed. Amplification for M. synoviae DNA was done as per OIE, (2008).
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