pVI110 locus of insertion were confirmed for the 8 loss-of-function mutant as described by17 (link), with the following modifications. Genomic DNA was digested sequentially with ClaI and BstBI restriction enzymes (NEB). Digested fragments were ligated using T4 DNA ligase (NEB) accordingly to manufacture’s instructions. Products of ligation were transformed into E. coli TG1 thermo-competent cells, in which circularized fragments containing the transposon behave as plasmids. Plasmids were isolated and sequenced (Genewiz) with the primers OLB221 and OLB215 (Supplementary Table 3 ). Identification of transposon target sequences was performed with the BLAST software from the National Center for Biotechnology Information (NCBI).
Bstbi restriction enzyme
Manufactured by New England Biolabs
Sourced in United States
BstBI is a type II restriction enzyme that recognizes and cleaves the DNA sequence 5'-TTCGAA-3'. It is a useful tool for molecular biology research, DNA cloning, and genetic engineering applications.
Automatically generated - may contain errors
4 protocols using bstbi restriction enzyme
1
Transposon Insertion Site Identification
2
Transposon Insertion Site Identification
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pVI110 locus of insertion were confirmed for the 8 loss-of-function mutant as described by17 (link), with the following modifications. Genomic DNA was digested sequentially with ClaI and BstBI restriction enzymes (NEB). Digested fragments were ligated using T4 DNA ligase (NEB) accordingly to manufacture’s instructions. Products of ligation were transformed into E. coli TG1 thermo-competent cells, in which circularized fragments containing the transposon behave as plasmids. Plasmids were isolated and sequenced (Genewiz) with the primers OLB221 and OLB215 (Supplementary Table 3 ). Identification of transposon target sequences was performed with the BLAST software from the National Center for Biotechnology Information (NCBI).
3
Cloning MRE11 Mutants into pBABE-Puro-Halo
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dsDNA molecules (gBlocks Gene Fragments) were ordered from IDT to design DNA constructs with the BstBI region added to the human MRE11(H129N) or DB1 and DB2 deletion fragment. The gBlocks and pBABE-Puro-Halo vector were then cut using the BstBI restriction enzyme (NEB, R0519S). Samples were resuspended in nuclease-free distilled water (IDT, 11-05-01-04) to a concentration of 10 ng µl–1. Samples were heated at 50 °C for 20 min per the manufacturer’s instructions and stored at −20 °C. Finally, the MRE11(H129N) and MRE11(DB1Δ/DB2Δ) sequences were inserted into the pBABE-Puro-Halo vector using Quick DNA ligase (NEB, M2200S).
4
MITF Transcriptional Regulation of Melanogenic Genes
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A yeast one-hybrid (Y1H) assay was performed using the MatchmakerTM Gold Yeast One-Hybrid System (Clontech, USA) as described in the manufacturer's protocol. Briefly, the promoter sequences of MpPO/MpCTSK/MpBCL-2 were cloned into the pAbAi vector, which contains an AbA resistance (AbAr) reporter gene, to construct CTSK/PO/BCL-2-pAbAi. Then, the CTSK/PO/BCL-2-pAbAi plasmid was linearized using the BstBI restriction enzyme (NEB, USA) and integrated into the genome of the Y1HGold yeast strain. The minimal inhibitory concentration of Aureobasidin A (AbA) for the yeast strain was confirmed using 300 ng/ml. Additionally, the full coding sequence of MpMITF was cloned into the pGADT7 vector to construct MITF-pGADT7, which expressed MITF fusion proteins containing the yeast GAL4 transcriptional activation domain (AD). The MITF-pGADT7 vector was transformed into the yeast strain containing CTSK/PO/BCL-2-pAbAi and screened on SD/-Leu/AbA plates. When the MITF protein binds to the MpPO/MpCTSK/MpBCL-2 promoter sequences, the GAL4 AD will activate expression of AbAr, allowing the cells to grow on media containing the AbA antibiotic.
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