Escherichia coli K-12 strains MG1655 was used for directed evolution and degradation mechanism investigation. RARE [4 (link)] strain was used for in vivo fermentation. Random mutagenesis of AD by error-prone PCR was performed with the GeneMorphII Random Mutagenesis Kit (Agilent Technologies) with initial template amount 100 ng, PCR cycle number 25 to achieve low mutation frequency (0–4.5%). ADs were cloned into pZa23MCS without codon optimization. Plasmids and DNA were purified using Monarch kits (NEB) according the manufacturer’s instructions. Restriction digests were carried out using standard protocols of NEB restriction endonucleases. For ligation of DNA fragments, T4 DNA ligase (Life Technologies) was used according to manufacturer’s instructions. Gene deletions were performed by red-mediated recombination [45 (link)]. The I-TASSER Suite was employed to generate structural model of ADs minus degrons [46 (link)]. Autodock Vina was used for molecular docking [47 (link)]. Protein stability was predicted by SCooP [48 (link)]. GRAMMX server was used for protein–protein interaction prediction [49 (link)].
Monarch kits
Manufactured by New England Biolabs
The Monarch kits are a line of laboratory reagents and tools designed for nucleic acid purification and manipulation. The kits provide a straightforward and efficient method for extracting and purifying DNA or RNA from various sample types. The core function of the Monarch kits is to enable the isolation and concentration of nucleic acids for downstream applications such as PCR, sequencing, or further analysis.
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Lab products found in correlation
3 protocols using monarch kits
1
Directed Evolution of Enzyme Degradation
2
Directed evolution of adenylate deaminase
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Escherichia coli K-12 strains MG1655 was used for directed evolution and degradation mechanism investigation. RARE [4] strain was used for in vivo fermentation. Random mutagenesis of AD by errorprone PCR was performed with the GeneMorphII Random Mutagenesis Kit (Agilent Technologies) with initial template amount 100 ng, PCR cycle number 25 to achieve low mutation frequency (0-4.5%). ADs were cloned into pZa23MCS without codon optimization. Plasmids and DNA was puri ed using Monarch kits (NEB) according the manufacturer's instructions. Restriction digests were carried out using standard protocols of NEB Restriction Endonucleases. For ligation of DNA fragments, T4 DNA ligase (Life Technologies) was used according to manufacturer's instructions. Gene deletions were performed by-red mediated recombination [45] . The I-TASSER Suite was employed to generate structural model of ADs minus degrons [46] . Autodock Vina was used for molecular docking [47] . Protein stability was predicted by SCooP [48] . GRAMMX server was used for protein-protein interaction prediction [49] .
3
Fine-Mapping the AET1 Locus in Rice
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The aet1 mutant was crossed with a japonica variety, Jiahua-1, and the F 1 plants were self-pollinated to generate an F 2 mapping population. To finemap the AET1 locus, we developed new molecular markers by screening for predicted simple sequence repeats and identified new cleaved amplified polymorphism sequence markers. AET1 was mapped to a 23.3-kb region on the long arm of chromosome 5, and all DNA fragments from this region were amplified from both aet1 and wild-type genomic DNA by PCR for DNA sequencing. PCR amplifications were performed using 23 PCR mix (Tiangen #KT207). DNA purification and plasmid DNA extraction were performed using Monarch kits from New England Biolabs (cat. #T1020L and #1010L). The mapping information is shown in Figure 1B, and the PCR primer pairs are listed in Supplemental Table 2.
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