Maxima Reverse Transcriptase (100U; Thermo Fisher Scientific) was used to reverse transcribe 20 ng RNA with both oligo(dT) and random hexamers in a 10 μl reaction system. Next, 0.5 μl cDNA was used in 10 μl PCR reactions based on Phire Hot Start II PCR Master Mix (Thermo Fisher Scientific) and primers (0.5 μM each), and amplified using a touchdown program with either 15 or 70 s extension step for short or long amplification, respectively. The PCR products were examined on 0.8–1.2% agarose gels (Thermo Fisher Scientific). Primer sequences are provided in Supplementary Table 3 .
Phire hot start 2 pcr master mix
Manufactured by Thermo Fisher Scientific
Phire Hot Start II PCR Master Mix is a ready-to-use solution for PCR amplification. It contains all the necessary components, including Phire Hot Start II DNA Polymerase, dNTPs, and buffer, to perform PCR reactions.
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Lab products found in correlation
4 protocols using phire hot start 2 pcr master mix
1
Reverse Transcription and Touchdown PCR
2
Multilocus Sequence Typing of Wolbachia
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Five conserved genes (
gatB,
coxA,
hcpA,
ftsZ and
fbpA) were amplified to determine any newly discovered
Wolbachia strains as previously described with the use of M13 adaptors or degenerate primers
46 (link)
. MLST PCRs consisted of 10 µL of Phire Hot Start II PCR Master Mix (Thermo Scientific), primers with a final concentration of 1 µM, 1 µL of PCR-grade water and 2 µL template cDNA (20 µL total). PCR cycling was carried out in a Bio-Rad T100 Thermal Cycler using cycling conditions that were optimised for different MLST genes tested with the Phire Hot Start II PCR Master Mix. Three genes (
gatB,
hcpA and
fbpA genes) had the following cycling: 98°C for 30 sec, 34 cycles of 98°C for 5 sec, 65°C for 5 sec, 72°C for 10 sec, 72°C for 1 min. For two genes (
coxA and
ftsZ) cycling was: 98°C for 30 sec, 34 cycles of 98°C for 5 sec, 55°C for 5 sec and 72°C for 30 sec, 72°C for 1 min.
gatB,
coxA,
hcpA,
ftsZ and
fbpA) were amplified to determine any newly discovered
Wolbachia strains as previously described with the use of M13 adaptors or degenerate primers
46 (link)
. MLST PCRs consisted of 10 µL of Phire Hot Start II PCR Master Mix (Thermo Scientific), primers with a final concentration of 1 µM, 1 µL of PCR-grade water and 2 µL template cDNA (20 µL total). PCR cycling was carried out in a Bio-Rad T100 Thermal Cycler using cycling conditions that were optimised for different MLST genes tested with the Phire Hot Start II PCR Master Mix. Three genes (
gatB,
hcpA and
fbpA genes) had the following cycling: 98°C for 30 sec, 34 cycles of 98°C for 5 sec, 65°C for 5 sec, 72°C for 10 sec, 72°C for 1 min. For two genes (
coxA and
ftsZ) cycling was: 98°C for 30 sec, 34 cycles of 98°C for 5 sec, 55°C for 5 sec and 72°C for 30 sec, 72°C for 1 min.
3
Multilocus Sequence Typing of Wolbachia
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Five conserved genes (
gatB,
coxA,
hcpA,
ftsZ and
fbpA) were amplified to determine any newly discovered
Wolbachia strains as previously described with the use of M13 adaptors or degenerate primers
46 (link)
. MLST PCRs consisted of 10 µL of Phire Hot Start II PCR Master Mix (Thermo Scientific), primers with a final concentration of 1 µM, 1 µL of PCR-grade water and 2 µL template cDNA (20 µL total). PCR cycling was carried out in a Bio-Rad T100 Thermal Cycler using cycling conditions that were optimised for different MLST genes tested with the Phire Hot Start II PCR Master Mix. Three genes (
gatB,
hcpA and
fbpA genes) had the following cycling: 98°C for 30 sec, 34 cycles of 98°C for 5 sec, 65°C for 5 sec, 72°C for 10 sec, 72°C for 1 min. For two genes (
coxA and
ftsZ) cycling was: 98°C for 30 sec, 34 cycles of 98°C for 5 sec, 55°C for 5 sec and 72°C for 30 sec, 72°C for 1 min.
gatB,
coxA,
hcpA,
ftsZ and
fbpA) were amplified to determine any newly discovered
Wolbachia strains as previously described with the use of M13 adaptors or degenerate primers
46 (link)
. MLST PCRs consisted of 10 µL of Phire Hot Start II PCR Master Mix (Thermo Scientific), primers with a final concentration of 1 µM, 1 µL of PCR-grade water and 2 µL template cDNA (20 µL total). PCR cycling was carried out in a Bio-Rad T100 Thermal Cycler using cycling conditions that were optimised for different MLST genes tested with the Phire Hot Start II PCR Master Mix. Three genes (
gatB,
hcpA and
fbpA genes) had the following cycling: 98°C for 30 sec, 34 cycles of 98°C for 5 sec, 65°C for 5 sec, 72°C for 10 sec, 72°C for 1 min. For two genes (
coxA and
ftsZ) cycling was: 98°C for 30 sec, 34 cycles of 98°C for 5 sec, 55°C for 5 sec and 72°C for 30 sec, 72°C for 1 min.
4
Gateway Cloning for Plasmid Construction
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All the plasmids in this study were constructed using the modified Gateway cloning protocol (Invitrogen, California, USA) published previously (Kovalchuk et al., 2012 ). 5‐′ and 3‐′ fragments for the deletion cassette were amplified with Phire Hot Start II PCR Master Mix (Thermo Fisher Scientific, San Jose, CA) using specific primers and cloned into corresponded Gateway donor vectors pDONR P4‐P1R and pDONR P2R‐P3, respectively, using BP clonase II enzyme mix (Invitrogen). The resulting plasmids were purified from kanamycin‐resistant E. coli Dh5α transformants and subsequently recombined with the Gateway destination vector pDEST R4‐R3 and pDONR221‐AMDS for in vitro recombination using LR clonase II enzyme mix. For expression, the modified pDONR221‐AMDS plasmid was used. In this construct the pcbC (isopenicillin synthase) promoter region was ligated downstream of the amdS gene. After incubation, the reaction mixture was transformed to E. coli Dh5α and the final plasmids were isolated from the ampicillin‐resistant transformants (Salo et al., 2016 ).
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