Gateway recombinational cloning (Thermo Fisher Scientific) was used to make an untagged rhinovirus 3C protease expression vector for co-lysis experiments. The 3C protease ORF was amplified by PCR from pET/3C using primers PE-2856 and PE-2857 (Table 1 ). These primers incorporated the attB recombination sites along with an appropriately positioned ribosome-binding site (RBS) in the PCR product. Subsequently, the PCR product was used in a BP reaction with pDONR221 (Thermo Fisher Scientific) to generate the entry clone pSRK2703. The entry clone was recombined in an LR reaction with pDEST42 (Thermo Fisher Scientific) to generate the IPTG-inducible 3C protease expression vector pSRK2706 (Table 2 ).
Pdest42
Manufactured by Thermo Fisher Scientific
Sourced in United Kingdom
The PDEST42 is a laboratory equipment product offered by Thermo Fisher Scientific. It is designed to perform a core function, but a detailed and unbiased description cannot be provided without the risk of extrapolation or interpretation.
Automatically generated - may contain errors
3 protocols using pdest42
1
Recombinant Rhinovirus 3C Protease Expression
2
Cloning and Expression of Slug Gut Metagenome Gene
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Gene_id_40363 was amplified from the slug’s gut metagenomic DNA using gene-specific primers—FP: CACCATGAAACATGACCAC, RP: GCCTCATCGAAATAGTGC—and the following PCR conditions: initial denaturation at 98 °C for 1 min; 35 cycles of denaturation at 98 °C for 40 s, an annealing temperature of 61 °C for 30 s, and extension for 19 s at 72 °C; and a final extension time of 10 min. PCR amplification was conducted with a high-fidelity thermostable DNA polymerase Q5 (NEB, Hitchin, UK) in a 25 µL reaction containing 12.5 µL of Q5 Hi-Fidelity 2X Master Mix, 1.25 µL of (10 µM forward and 10 µM reverse primers), and ~60 ng of template DNA.
The PCR-amplified product (654 bp) was cloned into an entry vector pENTR/SD/D/TOPO (ThermoFischer Scientific Leicester, UK) following the manufacturer’s protocol. To generate the recombinant expression vector (Figure 8 ), pENTR: Gene_id_40363 was recombined with the destination vector pDEST42 (ThermoFischer Scientific, Leicester, UK) following the manufacturer’s protocol. The recombinant expression vector was used to transform Escherichia coli (E. coli) DE3 cells. The desired transformant was screened with restriction digest and sequencing.
The PCR-amplified product (654 bp) was cloned into an entry vector pENTR/SD/D/TOPO (ThermoFischer Scientific Leicester, UK) following the manufacturer’s protocol. To generate the recombinant expression vector (
3
Recombinant Rhinovirus 3C Protease Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Gateway recombinational cloning (Thermo Fisher Scientific) was used to make an untagged rhinovirus 3C protease expression vector for co-lysis experiments. The 3C protease ORF was amplified by PCR from pET/3C using primers PE-2856 and PE-2857 (Table 1 ). These primers incorporated the attB recombination sites along with an appropriately positioned ribosome-binding site (RBS) in the PCR product. Subsequently, the PCR product was used in a BP reaction with pDONR221 (Thermo Fisher Scientific) to generate the entry clone pSRK2703. The entry clone was recombined in an LR reaction with pDEST42 (Thermo Fisher Scientific) to generate the IPTG (isopropyl β-d -1-thiogalactopyranoside)-inducible 3C protease expression vector pSRK2706 (Table 2 ).
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