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Mycycler gradient cycler

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The MyCycler gradient cycler is a thermal cycler used for performing polymerase chain reaction (PCR) experiments. It features a gradient function, which allows for the optimization of primer annealing temperatures across multiple samples simultaneously.

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Lab products found in correlation

E coli top10 cells, by Thermo Fisher Scientific (2 mentions) Ptrchisa vector, by Thermo Fisher Scientific (2 mentions) Gibson assembly master mix, by New England Biolabs (2 mentions) Q5 high fidelity 2 master mix, by New England Biolabs (2 mentions)

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MyCycler Gradient MyCycler Bio-Rad cyclers

4 protocols using mycycler gradient cycler

1

Cloning and Characterization of clbH Gene

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Linearized pTrcHisA vector was PCR amplified from pTrcHisA vector (Invitrogen) using primers shown in Supplementary Table 4. Full-length clbH, truncated clbH (clbH-C-A2-PCP), and two adjacent genes clbH and clbI (clbH-clbI) were PCR amplified from E. coli CFT073 genomic DNA (ATCC) using primers shown in Supplementary Table 4. PCR reactions (20 μL) contained 10 μL Q5 High-Fidelity 2× Master Mix (New England Biolabs), 1 ng of DNA template, and 500 pmoles of each primer. Thermocycling was carried out in a MyCycler gradient cycler (Bio-rad) using the following condition: denaturation for 1 min at 98 °C, followed by 35 cycles of 10 sec at 98 °C, 30 sec at 72 °C, 3 min for clbH (2 min for clbH-C-A2-PCP and 4 min for clbH-clbI) at 72 °C, and a final extension of 5 min at 72 °C.
Gibson assembly reactions (10 μL) contained 100 ng linearized pTrcHisA vector, 3-fold of molar excess purified PCR products of clbH, clbH-C-A2-PCP, or clbH-clbI, and 5 μL of 2× Gibson Assembly Master Mix (New England Biolabs). The mixtures were incubated at 50 °C for 15 min and used to transform 50 μL of chemically competent E. coli TOP10 cells (Invitrogen). The identity of the assembled plasmids was confirmed by sequencing.
The pTrcHisA-clbH, -clbH-C-A2-PCP, and -clbH-clbI plasmids were electroporated into electrocompetent E. coli DH10B BACpksΔclbDtoMΔclbOtoQ and stored at −80 °C as frozen LB/glycerol stocks.
Zha L., Jiang Y., Henke M.T., Wilson M.R., Wang J.X., Kelleher N.L, & Balskus E.P. (2017). Colibactin assembly line enzymes use S-adenosylmethionine to build a cyclopropane ring. Nature chemical biology, 13(10), 1063-1065.
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2

Cloning and Characterization of clbH Gene

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Linearized pTrcHisA vector was PCR amplified from pTrcHisA vector (Invitrogen) using primers shown in Supplementary Table 4. Full-length clbH, truncated clbH (clbH-C-A2-PCP), and two adjacent genes clbH and clbI (clbH-clbI) were PCR amplified from E. coli CFT073 genomic DNA (ATCC) using primers shown in Supplementary Table 4. PCR reactions (20 μL) contained 10 μL Q5 High-Fidelity 2× Master Mix (New England Biolabs), 1 ng of DNA template, and 500 pmoles of each primer. Thermocycling was carried out in a MyCycler gradient cycler (Bio-rad) using the following condition: denaturation for 1 min at 98 °C, followed by 35 cycles of 10 sec at 98 °C, 30 sec at 72 °C, 3 min for clbH (2 min for clbH-C-A2-PCP and 4 min for clbH-clbI) at 72 °C, and a final extension of 5 min at 72 °C.
Gibson assembly reactions (10 μL) contained 100 ng linearized pTrcHisA vector, 3-fold of molar excess purified PCR products of clbH, clbH-C-A2-PCP, or clbH-clbI, and 5 μL of 2× Gibson Assembly Master Mix (New England Biolabs). The mixtures were incubated at 50 °C for 15 min and used to transform 50 μL of chemically competent E. coli TOP10 cells (Invitrogen). The identity of the assembled plasmids was confirmed by sequencing.
The pTrcHisA-clbH, -clbH-C-A2-PCP, and -clbH-clbI plasmids were electroporated into electrocompetent E. coli DH10B BACpksΔclbDtoMΔclbOtoQ and stored at −80 °C as frozen LB/glycerol stocks.
Zha L., Jiang Y., Henke M.T., Wilson M.R., Wang J.X., Kelleher N.L, & Balskus E.P. (2017). Colibactin assembly line enzymes use S-adenosylmethionine to build a cyclopropane ring. Nature chemical biology, 13(10), 1063-1065.
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3

Mutagenesis of SznF using Quikchange Protocol

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The SznF point mutants were constructed by adapting the Quikchange protocol33 (link). In brief, each PCR reaction contained 41.65 μL H2O, 1.5 μL DMSO, 0.2 μL dNTP (50 mM stock), 0.4 μL of pET28a-SznF (50 ng/μL stock), 0.125 μL of each mutagenesis primer (200 μM stock, nucleotide sequences in Supplementary Table 6), 1 μL of Pfu Turbo AD and 5 μL of Buffer (Turbo AD). Thermocycling was carried out in a MyCycler gradient cycler (Bio-Rad) using the following parameters: denaturation for 2 min at 95 ºC; 18 cycles of 0.5 min at 95 ºC, 1 min at 71 ºC, and an extension time of 13 min at 72 ºC. 40 μL of the PCR reaction mixture was digested with 2 μL of DpnI for 2 h at 37 ºC. Afterwards, the reaction was transformed into chemically competent E. coli Top10 cells, and the constructs containing the desired point mutants were confirmed by sequencing. The mutant SznF enzymes were expressed, purified, and assays using the procedures described above for wild type SznF.
Ng T.L., Rohac R., Mitchell A., Boal A.K, & Balskus E.P. (2019). An N-nitrosating metalloenzyme constructs the pharmacophore of streptozotocin. Nature, 566(7742), 94-99.
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4

Mutagenesis of SznF using Quikchange Protocol

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The SznF point mutants were constructed by adapting the Quikchange protocol33 (link). In brief, each PCR reaction contained 41.65 μL H2O, 1.5 μL DMSO, 0.2 μL dNTP (50 mM stock), 0.4 μL of pET28a-SznF (50 ng/μL stock), 0.125 μL of each mutagenesis primer (200 μM stock, nucleotide sequences in Supplementary Table 6), 1 μL of Pfu Turbo AD and 5 μL of Buffer (Turbo AD). Thermocycling was carried out in a MyCycler gradient cycler (Bio-Rad) using the following parameters: denaturation for 2 min at 95 ºC; 18 cycles of 0.5 min at 95 ºC, 1 min at 71 ºC, and an extension time of 13 min at 72 ºC. 40 μL of the PCR reaction mixture was digested with 2 μL of DpnI for 2 h at 37 ºC. Afterwards, the reaction was transformed into chemically competent E. coli Top10 cells, and the constructs containing the desired point mutants were confirmed by sequencing. The mutant SznF enzymes were expressed, purified, and assays using the procedures described above for wild type SznF.
Ng T.L., Rohac R., Mitchell A., Boal A.K, & Balskus E.P. (2019). An N-nitrosating metalloenzyme constructs the pharmacophore of streptozotocin. Nature, 566(7742), 94-99.
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