FLAG‐L166P DJ‐1, FLAG‐C106S DJ‐1 plasmids, and anti‐DJ‐1 polyclonal antibody were kindly supplied by Dr. Hiroyoshi Ariga, Graduate School of Pharmaceutical Sciences, Hokkaido University. Dehydrated minimal essential medium (MEM) and F‐12 medium were purchased from Gibco. Lipofectin was from Invitrogen. FastDigest Xho I and FastDigest EcoR I were from Fermentas. Endotoxin‐free plasmid preparation kit was from BioTeke. The SH‐SY5Y cell line was from the Cell Bank of the Chinese Academy of Medical Sciences. BCA protein assay reagent kit was from Pierce. PVDF membranes were from Millipore. Anti‐FLAG polyclonal antibody was from GeneTex. Cistanche extracts, including acteoside, echinacoside, caffeic acid, and Cistanche total glycosides, were supplied by the Department of Natural Medicines, School of Pharmaceutical Sciences, Peking University.
Fastdigest ecori
Manufactured by Thermo Fisher Scientific
Sourced in United States
FastDigest EcoRI is a restriction enzyme that recognizes and cleaves the DNA sequence 5'-GAATTC-3'. It is designed to provide rapid and efficient DNA digestion for various molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Fastdigest hindiii, by Thermo Fisher Scientific (2 mentions)
T4 dna ligase, by New England Biolabs (2 mentions)
T4 dna ligase, by Thermo Fisher Scientific (2 mentions)
Fastdigest xhoi, by Thermo Fisher Scientific (1 mentions)
Lipofectin, by Thermo Fisher Scientific (1 mentions)
F12 medium, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Seqman tool, by DNASTAR (1 mentions)
Specific primers, by Merck Group (1 mentions)
Fast alkaline phosphatase, by Thermo Fisher Scientific (1 mentions)
T4 dna ligase enzyme, by New England Biolabs (1 mentions)
Zymoclean gel dna recovery kit, by Zymo Research (1 mentions)
Miniprep kit, by Qiagen (1 mentions)
Dneasy blood tissue extraction kit, by Qiagen (1 mentions)
E coli dh5 alpha competent cells, by New England Biolabs (1 mentions)
Gotaq hot start green master mix, by Promega (1 mentions)
Plvx ef1α ires mcherry, by Takara Bio (1 mentions)
Fastdigest bamhi, by Thermo Fisher Scientific (1 mentions)
Gblock gene fragment, by Integrated DNA Technologies (1 mentions)
Rnase a solution, by Qiagen (1 mentions)
14 protocols using fastdigest ecori
1
Characterization of Mutant DJ-1 Proteins
2
Cloning and Characterization of Eaf2 CDS
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The Eaf2 coding sequence (CDS) was amplified by polymerase chain reaction (PCR) using murine cDNA, which was reverse transcribed from total RNA extracted from the rat cells. This was maintained in our laboratory as a template. The primers used were as follows: Eaf2-CDS forward, 5′-GTATGAAAGCTTAAGGCCAAAAGCGG-3′ and reverse, 5′-GCCCGAATTCATCTCACAAATGTTTTCTCTGT-3′. The primers were synthesized by Sangon Biotech (Shanghai, China). The PCR was performed using a Life Express PCR Instrument (Bioer, Hangzhou, China) and the following cycling conditions were used: 95 °C for 5 min; 95°C for 30 sec, 55°C for 30 sec, 72°C for 60 sec, 30 cycles; 72°C for 5 min and 4°C for 2 min. The products were purified using a multifunctional DNA purification kit (BioTeke, Beijing, China). The sequencing was performed by Sangon Biotech). The coding sequence was inserted into the p enhanced green fluorescence protein-N1 vector (Clontech, Mountain View, CA, USA) by the restriction enzymes FastDigest HindIII and FastDigest EcoRI (Fermentas, Burlington, CA, USA). The vector was sequenced for verification and was named as Eaf2 OE.
3
Genome Walking for Full-length phaC DNA
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Genome walking with inverse affinity nested PCR (IAN-PCR) was carried out to obtain the full-length phaC DNA sequences [61 (link)]. WGA seawater DNA was digested with FastDigest EcoRI (Fermentas, Lithuania) and self-ligated using a DNA Ligation Kit Ver1.2 (TaKaRa, Japan). For each genetic group, two sets of primers (for two rounds of amplification) (Additional file 6 : Table S1) were designed based on the partial phaC sequences obtained in the previous step. The first round of amplification (inverse affinity PCR) included a 5′-biotinylated primer that enabled the product to be purified using the KiloBase Binder Kit (Invitrogen, USA) and serve as a template for the second round of amplification (nested PCR). DNA sequences were determined by performing the same cloning and sequencing steps described in the previous section. Full-length phaC sequences were predicted in silico by assembling the genome walking DNA fragments using the SeqMan tool (DNASTAR, USA).
4
Metagenomic DNA Plasmid Construction
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The plasmids were prepared by amplifying the gene encoding the metagenomic DNA of the samples extracted for the sequences available in the LBMP55 . The following specific primers (Sigma-Aldrich) were synthesized using the commercial PCRBIO Ultra Mix 2 × Kit26 (link): forward 5'-TATAgaattcTTCGGACGCAATCCAGACACCAATCC-3', which contains the restriction site EcoRI, and reverse 3'-TATAaagcttTTACTTGAACAGCAATTGAG-5', which contains the restriction sites EcoRI and HindIII. The fragment corresponding to the gene was purified by the Zymoclean Gel DNA Recovery Kit from ZymoResearch. The vector pET28a ( +) (INVITROGEN) and insert were restricted with restriction enzymes (FastDigest EcoRI, Thermo Scientific; and FastDigest HindIII, Thermo Scientific) and dephosphorylated with the enzyme Fast Alkaline Phosphatase (1 U/µL, Thermo Scientific). The connection between the insert and the vector was performed according to the protocol of Sambrook and Russel56 (link) using the T4 DNA ligase enzyme (New England Biolabs) in a 3:1 ratio (insert: vector).
5
Fetal Tissue DNA Sequencing
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Recipient sheep were euthanized and fetuses collected at day 75 or 120 of gestation. Samples collected from different tissues were used for genomic DNA extraction using the DNeasy Blood & Tissue extraction kit (QIAGEN). PCR was performed using GoTaq Hot Start Green Master Mix (Promega) using the same primers and conditions described above. PCR products were purified, cloned into pCR™TOPO®TA vector (Life Technologies) and transformed into E. Coli DH5-alpha competent cells (NEB). Ten colonies were picked, cultured in LB broth, and plasmid DNA was extracted using a Miniprep kit (QIAGEN). Fast digest EcoRI (Thermo Scientific) was used to identify the positive colonies and samples were sent for Sanger sequencing (Quintarabio). Sequencing analysis was performed as described above.
6
CRISPR-Cas9 Transfection Efficiency Assay
7
Generation of PARK2 Lentiviral Plasmids
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PARK2 and PARK2-C431S lentiviral plasmids were generated by sub-cloning the human wild-type (from plasmid pRK5-HA-Parkin; cat. no. 17613; Addgene, Inc.) or the catalytically inactive C431S mutant PARK2 (from pEGFP-parkin C431S; cat. no. 45877; Addgene, Inc.) into the restriction enzyme sites FastDigest EcoRI (Thermo Fisher Scientific, Inc.) and FastDigest BamHI (Thermo Fisher Scientific, Inc.) of plasmid pLVX-EF1α-IRES-mCherry (Takara Bio USA, Inc.).
8
Engineered dCas9-MYB Fusion Protein
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We modified the vector pX330A_dCas9–1 × 4 (a gift from Takashi Yamamoto, Addgene plasmid #63598) by inserting a gBlock Gene Fragment (Integrated DNA Technologies) encoding the MYB TAD followed by a p2A signal [101 (link)] and mCherry after the dCas9 ORF. The resulting vector expresses a dCas9-MYB fusion and mCherry as separate proteins from a single mRNA transcript. The vector and gBlock were digested with FseI (New England BioLabs) and FastDigest EcoRI (ThermoFisher Scientific) and ligated using T4 DNA Ligase (New England BioLabs). We named this new vector pX330g_dCas9-MYB. SgRNAs used in the study (Supplementary Table S3 ) were designed using the CRISPR design tool at crispr.mit.edu. DNA oligos were synthesized with BbsI overhangs for cloning into pX330g_dCas9-MYB (Integrative DNA Technology). Drop-in of sgRNAs followed the cloning protocol described in Cong et al. [102 (link)].
9
DNA Extraction and Restriction Digest
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Isolation of total DNA was performed via the method employed by Lee et al., with minor changes1 (link),28 (link),29 (link). Overnight cultures harboring plasmids of interest were inoculated 1:100 in LB kanamycin. Cultures were grown to an OD600 of ~0.3, and then 2 mL of culture was pelleted and frozen at –80 °C. Cell pellets were resuspended in 400 μL of 50 mM Tris/50 mM EDTA, pH 8, and then permeabilized with 8 μL of 50 mg/mL lysozyme (Sigma Aldrich, USA) in 10 mM Tris/1 mM EDTA, pH 8, followed by incubation at 37 °C for 30 minutes. Cells were then lysed by the addition of 8 μL of 5% SDS and 8 μL of 20 mg/mL Proteinase K solution (Qiagen, USA), after which they were mixed with a syringe with a 21-gauge, 1.5-inch needle, followed by incubation at 50 °C for 30 minutes. After inactivating Proteinase K by incubation at 75 °C for 10 min, 2 μL of 100 mg/mL RNase A solution (Qiagen, USA) was added and then incubated at 37 °C for 30 minutes. Total DNA was extracted via a phenol:chloroform extraction, followed by ethanol precipitation as described previously1 (link). Isolated gDNA was digested for 6 hours at 37 °C with Fast Digest EcoRI (Thermo Fisher Scientific, Waltham, MA).
10
Amplification and Analysis of Viral Genome Region
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The virus genome region containing the EcoRI site in the mutant 47832EcoRI was amplified by RT-PCR using primers as listed in Supplementary Results, Table S1 , followed by restriction enzyme analysis. Briefly, RNA was isolated from supernatants of infected cells using the QIAamp viral RNA mini kit (Qiagen, Hilden, Germany), and the QIAGEN OneStep RT-PCR Kit (Qiagen, Hilden, Germany) was used for RT-PCR. The PCR products were purified using the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany). Thereafter, the FastDigest EcoRI (Thermo Fisher Scientific, Waltham, MA, USA) was added together with the FastDigest buffer (Thermo Fisher Scientific, Waltham, MA, USA) and incubated at 37 °C for 15 min. The products were analyzed by electrophoresis on ethidium bromide-stained agarose gels.
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