A catalytic mutant of HAUSP (C223S) and ANXA1 mutants (S5A, S182A, S5A/S182A) were generated by site-direct mutagenesis. The following primer sequences were used for generating mutants: ANXA1 (S5A); forward, 5′-ATG GCA ATG GTA GCA GAA TTC CTC A-3′, reverse, 5′-TGA GGA ATT CTG CTA CCA TTG CCA T-3′, ANXA1 (S182A); forward, 5′-CGG AAC GCT TTG CTT GCT CTT GCT AAG GGT G-3′, reverse, 5′-CAC CCT TAG CAA GAG CAA GCA AAG CGT TCC G-3′. ANXA1 mutant (S5A/S182A) was generated using ANXA1 (S5A) as a template and ANXA1 (S182A) primer. After PCR and gel purification, Dpn I (Enzynomics, Daejeon, Korea) enzyme was added.
Dpn 1
Manufactured by Enzynomics
Dpn I is a restriction enzyme that recognizes and cleaves the DNA sequence 5'-GATC-3'. It is commonly used in molecular biology applications to generate specific DNA fragments from plasmids or other DNA sources.
Automatically generated - may contain errors
Lab products found in correlation
Bamhi, by Enzynomics (1 mentions)
Pfu dna polymerase, by Thermo Fisher Scientific (1 mentions)
Plvx ires hyg vector, by Takara Bio (1 mentions)
Ampone taq premix, by GeneAll (1 mentions)
Histrap, by GE Healthcare (1 mentions)
Bis ans, by Thermo Fisher Scientific (1 mentions)
Npfu forte polymerase, by Enzynomics (1 mentions)
T4 polynucleotide kinase, by Enzynomics (1 mentions)
T4 ligase, by Enzynomics (1 mentions)
6 protocols using dpn 1
1
Generation of HAUSP and ANXA1 Mutants
2
Generating GFP-tagged Sur8 Overexpression Lentiviral Plasmids
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For generating GFP-tagged Sur8-overexpressing lentiviral plasmids, GFP was inserted into pLVX-IRES-Hyg vector (#632185, Clonetech, Palo Alto, CA) at NotI enzyme site prior to cloning Sur8. Human Sur8 complementary DNA (cDNA) was amplified by reverse transcription polymerase chain reaction (RT-PCR) using the following primers: forward 5′-ATAACCGGGATCCACCATGAGTAGTAGTTTAGGA-3′; reverse 5′-TCTAGAGGATCCGACCATGGCACGATATGG-3′. It was inserted into the pLVX-IRES-Hyg vector expressing GFP after digestion with BamHI (Enzynomics, Daejeon, Korea).
For site-directed mutagenesis, point mutations of Sur8 were introduced by PCR using Pfu DNA polymerase (Invitrogen, Carlsbad, CA). The mutagenic oligonucleotides used for mutagenesis are shown inSupplementary Table 1 . PCR reactions were run in the following condition: 15 cycles of 30 seconds at 95°C, then 1 minute at 54°C followed by 10 minutes at 68°C. The PCR products were digested with DpnI (Enzynomics) for 1 hour for removal of the template plasmids. All mutant plasmids were verified by DNA sequencing (Cosmogenetech).
For site-directed mutagenesis, point mutations of Sur8 were introduced by PCR using Pfu DNA polymerase (Invitrogen, Carlsbad, CA). The mutagenic oligonucleotides used for mutagenesis are shown in
3
Generating GPx3 Promoter Mutants
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Double mutations in GRE6 and GRE7 of the GPx3 promoter were generated by PCR-mediated site-directed mutagenesis. For single mutation of GRE6, the complementary primers contained a triple-base mismatch in GRE6 that converts TGT to CAG using the pNL1.1::GPx3 promoter-GRE (WT) as the template. For double mutations of GRE6 and GRE7, the complementary primers contained a triple-base mismatch in GRE7 that converts GTCC to ATAA using the pNL1.1::GPx3 promoter-GRE6 mutant as the template. Briefly, specific PCR was carried out in 20 μl mixtures containing 10 ng of plasmid DNA and 20 pmol of each primer using AmpONE™ Taq premix (GeneAll). PCR conditions were 95°C for 10 min, 30 cycles of 95°C for 1 min, annealing at 45°C for 1 min and 72°C for 5 min, followed by a final extension step at 72°C for 10 min. The PCR products of the single and double mutations were collected and purified, treated with DpnI (Enzynomics, Korea) to remove the original DNA, and then transformed into DH5α cells. The single and double mutations were confirmed by nucleotide sequencing after plasmid isolation (An et al., 2011 (link); 2015 (link)).
4
Recombinant Sphingomonas Protein Production
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Sphingomonas sp. PAMC 26621
was provided by the Polar and Alpine Microbial Collection
of the Korea Polar Research Institute (Incheon, South Korea).26 (link) The ectrx gene in the pET32
expression vector was obtained from Addgene (Watertown, MA). The pET28(+)
expression vector was acquired from Novagen (Madison, WI). The nPfu-Forte polymerase and Dpn I were obtained from Enzynomics
(Daejeon, South Korea). HisTrap and Capto Q columns were purchased
from GE Healthcare (Piscataway, NJ). Bis-ANS was purchased from Invitrogen
(Waltham, MA). DTNB (Ellman’s Reagent) was purchased from ThermoFisher
Scientific (Waltham, MA). All other chemical reagents were purchased
from Sigma (St. Louis, MO) or Tokyo Chemical Industry (Tokyo, Japan)
unless stated otherwise.
was provided by the Polar and Alpine Microbial Collection
of the Korea Polar Research Institute (Incheon, South Korea).26 (link) The ectrx gene in the pET32
expression vector was obtained from Addgene (Watertown, MA). The pET28(+)
expression vector was acquired from Novagen (Madison, WI). The nPfu-Forte polymerase and Dpn I were obtained from Enzynomics
(Daejeon, South Korea). HisTrap and Capto Q columns were purchased
from GE Healthcare (Piscataway, NJ). Bis-ANS was purchased from Invitrogen
(Waltham, MA). DTNB (Ellman’s Reagent) was purchased from ThermoFisher
Scientific (Waltham, MA). All other chemical reagents were purchased
from Sigma (St. Louis, MO) or Tokyo Chemical Industry (Tokyo, Japan)
unless stated otherwise.
5
Saturated Mutagenesis Library Construction
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Saturated mutagenesis was carried out using primers containing the NNK degenerate codon as described previously.51 (link) PCR was performed using custom-designed primers (Table S2† ). After Dpn I (Enzynomics) digestion for 1.5 h at 37 °C, the PCR mixtures were transformed to DH5α E. coli competent cells. Greater than 100 colonies were selected for each single-site randomized library, and they were inoculated to LB medium containing 50 mg L−1 kanamycin. After the overnight cell-growth at 37 °C, plasmids were extracted using a mini-prep kit and sent out for sequencing (Macrogen or Bionics). When the selected position was not fully randomized, additional PCRs with the redesigned primers were performed. Representative sequencing chromatograms for each library are shown in Fig. S1 in the ESI.†
6
Site-Saturation Mutagenesis of Sniper1 Codon
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For site saturation mutagenesis of the 1,007th codon in the Sniper1 sequence, the pBLC–Sniper1 plasmid was amplified using primers containing NNK (K = G or T) at the appropriate position (forward primer: agtaccccaagctggagagcnnkttcgtgtacggcgactacaagg; reverse primer: tcttgatcagggcggtgcc). PCR products were digested with DpnI (Enzynomics), treated with T4 polynucleotide kinase (Enzynomics) and ligated with T4 ligase (Enzynomics). The resulting product was transformed in DH5alpha cells. After Sanger sequencing of plasmids from 100 randomly selected colonies, variants containing 20 different amino acids at the 1,007th position were identified.
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