The oligonucleotide primers employed to prepare the mS100A8(C42S) variant by site-directed mutagenesis were synthesized by Integrated DNA Technologies (Coralville, IA). pET41a-mS100A8(C42S) was prepared using pET41a-mS100A8 as a template and a modified Quick-Change site-directed mutagenesis protocol. The forward primer 5′-GATGGTTACCACGGAGAGC CCGCAGTTCGTGCAG-3′ and the reverse primer 5′-CTGCACGAACTGCGGGCT CTCCGTGGTAACCATC-3′ (mutation sites underlined) were used in PCR reactions with Pfu Turbo DNA polymerase. The PCR protocol was 95 °C for 30 sec; 95 °C for 30 sec, 60 °C for 1 min, 68 °C for 12 min (25x); 4 °C hold temperature. DpnI (New England Biolabs) was used to degrade the template plasmid by adding a 0.75-μL aliquot to the sample, and incubating the sample at 37 °C for 3 h. A supplemental 0.75 μL aliquot of DpnI was added at 1.5 h into the incubation. The resulting DNA was transformed into chemically-competent E. coli TOP10 and the cells were plated on agar containing 50 μg/mL kanamycin. Single colonies were inoculated into LB (5 mL, 50 μg/mL kanamycin) and grown overnight, and plasmids were obtained using a miniprep kit (Qiagen). The plasmids were analyzed by DNA sequencing (Quintara Biosciences) to confirm the presence of the desired mutation and then transformed into chemically competent E. coli BL21(DE3) for protein overexpression.
Dpni
Manufactured by New England Biolabs
Sourced in United States, Germany, United Kingdom, China
DpnI is a type II restriction endonuclease enzyme that recognizes and cleaves the DNA sequence 5'-Gm6ATC-3' (where m6A represents N6-methyladenine). It is commonly used in molecular biology research for the removal of parental DNA template in site-directed mutagenesis and DNA assembly applications.
Automatically generated - may contain errors
Lab products found in correlation
T4 dna ligase, by New England Biolabs (35 mentions)
T4 polynucleotide kinase, by New England Biolabs (23 mentions)
Phusion high fidelity dna polymerase, by New England Biolabs (23 mentions)
Phusion polymerase, by New England Biolabs (18 mentions)
Qiaquick pcr purification kit, by Qiagen (17 mentions)
Phusion dna polymerase, by New England Biolabs (17 mentions)
Qiaprep spin miniprep kit, by Qiagen (16 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (13 mentions)
Pfu turbo dna polymerase, by Agilent Technologies (12 mentions)
Q5 polymerase, by New England Biolabs (10 mentions)
Q5 dna polymerase, by New England Biolabs (10 mentions)
T4 ligase, by New England Biolabs (9 mentions)
Primestar max dna polymerase, by Takara Bio (8 mentions)
Gibson assembly master mix, by New England Biolabs (8 mentions)
Phusion dna polymerase, by Thermo Fisher Scientific (7 mentions)
Miniprep kit, by Qiagen (7 mentions)
Cutsmart buffer, by New England Biolabs (7 mentions)
Pcr purification kit, by Qiagen (6 mentions)
T4 ligase buffer, by New England Biolabs (6 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (6 mentions)
474 protocols using dpni
1
Mutagenesis of mS100A8 Protein
2
Site-Directed Mutagenesis of PapB Variants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Each of the cluster Cys deletion variants of PapB was constructed using site-directed mutagenesis with nonoverlapping primers (Table S1 ). Standard protocol for PCR with Phusion DNA polymerase (NEB #M0530) was used to generate modified plasmids. Briefly, after PCR amplification using appropriate primers, the template DNA was digested with DpnI (NEB #R0176L) for 1 h at 37 °C to remove template DNA. An aliquot (10 μl) of the DpnI-treated sample was added to a mixture of 2 μl T4 DNA ligase buffer (NEB # B0202S), 1 μl of T4 DNA ligase (NEB #M0202S), 1 μl of T4 polynucleotide kinase (NEB #M0201S), and 6 μl of H2O. The mixture was incubated at 20 °C for 2 h to phosphorylate and ligate the new plasmid. The sequence of each construct was verified by sequencing at the University of Utah Core facilities. For each cluster deletion variant, all the putative coordinating Cys residues were mutated to Ala in the final constructs (RS: C119, C123, and C126; AC1: C352, C370 and C421; and AC2: C408, C411, C417, and C440).
3
Differential DNA Methylation Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chromosomal and plasmid DNA from dam-positive E. coli isolates A620b, cured C119 (cC119), U155, uropathogenic E. coli CFT073 and dam-positive control strain E. coli MG1655 were subjected to differential digestion for dam function with restriction endonucleases Sau3AI, MboI, and DpnI according to Chen et al. (61 (link)) with modifications. Essentially, 0.5 μg of chromosomal and plasmid DNA was digested for 1.5 h at 37°C with 2 U Sau3AI (Promega, WI, USA), 10 U DpnI (New England Biolabs, MA, USA), or 2.5 U MboI. Sau3AI cleaves DNA at GATC sites regardless of methylation state, DpnI cleaves GATC sites that have a methylated adenine residue, and MboI cleaves unmethylated GATC sites. The resulting DNA was visualized under UV on ethidium bromide-stained agarose gels.
4
Site-Directed Mutagenesis of ACE2 Plasmid
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Site-directed mutagenesis for the generation of plasmids expressing mutated pCEP4-myc-ACE2 was performed with the following primers: His378: 5′-ATGAGATGGGGTCAATCCAGTAT-3′ (forward) and 5′-ATACTGGATTGACCCCATCTCAT-3′ (reverse); Arg393: 5′-ACCTTTTCTGCTACAAAATGGAGCTAATGA-3′ (forward) and 5′-TCATTAGCTCCATTTTGTAGCAGAAAAGGT-3′ (reverse); His401: 5′-AAGGATTCTCAGAAGCTGTT-3′ (forward) and 5′-AACAGCTTCTGAGAATCCTT-3′ (reverse); Arg514: 5′-CTCATTCATTCAATATTACACAAGGA-3′ (forward) and 5′-TCCTTGTGTAATATTGAATGAATGAG-3′ (reverse). The reactions were performed with PCR kits (KAPA HiFi HotStart; KR0369; v10.17) on a PCR machine under the following conditions: 95°C for 3 min, 98°C for 20 s, 60°C for 15 s, and 72°C for 6 min; these three conditions were cycled 16 times; then 72°C for 7 min. The nonmutated PCR template was removed from the pool of PCR products by incubation with the restriction endonuclease Dpn I [R0176L, New England Biolabs (NEB)] at 37°C for 1 hour and then at 60°C for 20 min. The plasmids were circularized through a ligation reaction with T4 DNA ligase (M0202S) at room temperature for 2 hours before being used to transform 5-α Competent Escherichia coli (High Efficiency, NEB). All of the plasmids were then sequenced to verify the mutations.
5
Cloning tRNA variants into pBAD_sfGFP2am
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
tRNA variants were cloned into pBAD_sfGFP2am (10 (link),11 (link)) in the reverse direction (3′ to 5′). First the pBAD_sfGFP2am vector was opened through amplification of the plasmid around the tRNA. Following DpnI (New England Biolabs®) digest and cleanup to remove the methylated parent plasmid, the product was used as a template to insert the tRNA via two overlapping primers (W.M. Keck Foundation, Yale University). The first primer, in the reverse direction (3′ to 5′), had at least 15 bp complimentary to the rrcn terminator of pBAD_sfGFP2am and a tail containing a portion of the tRNA sequence (∼50 bp). The second primer, in the forward direction (5′ to 3′), had at least 20 bp complimentary to the 3′ end of the first primer, continued with the rest of the tRNA sequence, and then ended with at least 15 bp complimentary to the Lpp promoter of pBAD_sfGFP2am. NEBuilder HiFi (New England Biolabs®) was used to ensure complete vector assembly before transformation into DH5α cells for plasmid prep and sequencing (W.M. Keck Foundation, Yale University).
6
High-Fidelity DNA Amplification and Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All DNA fragments were amplified with PhusionR High-Fidelity DNA Polymerase (New England Biolabs, County Road Ipswich, MA, USA). PCR conditions and annealing temperatures were modified depending on primers and templates used in the reaction. PCR reactions using plasmid DNA as template were digested with DpnI (NEB, County Road Ipswich, MA, USA) for 1 h at 37 °C followed by inactivation at 65 °C for 20 min to lower background after transformation. PCR products were purified using QIAquick PCR Purification Kit (Qiagen GmbH, Strasse 1, Hilden, Germany). The DNA fragments for transformations were concentrated via ethanol precipitation to a final concentration of ~1 μg/μL, determined using NanoDrop2000 (Thermo Fisher Scientific, Waltham, MA, USA). The primers used are listed in Table S1 .
7
Generating Recombinant Vesicular Stomatitis Virus
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Mutations were introduced using a pair of self-complementary primers carrying the desired change. The cDNA clone was amplified for eighteen cycles using the high-fidelity Phusion DNA polymerase (New England Biolabs). To remove template DNA, amplification products were digested with DpnI (New England Biolabs), which selectively cuts methylated GATC sites. Products were used for transforming competent Escherichia coli cells by the rubidium chloride heat-shock method. Plasmid DNA was then purified using the Nucleospin Plasmid purification kit (Macherey-Nagel) and used for transfecting BHK-21 cells as previously described (Whelan et al. 1995;
Sanjuán, Moya, and Elena 2004 ). Briefly, young 90 per cent confluent BHK-21 cells were infected with a recombinant vaccinia virus expressing T7 RNA polymerase and then co-transfected with the full-length VSV cDNA clone and three helper plasmids encoding the P, L, and N proteins. Transfections were done using Lipofectamine LTX (Life Technologies), following manufacturer’s instructions. After 6 h, 25 µg/ml 1-β-D-arabinofuranosylcytosine was added to inhibit vaccinia replication. After 3–4 days, supernatants were tested for the presence of infectious VSV particles by the standard plaque assay, vaccinia virus was removed by filtration, and one additional VSV infection cycle was performed to reach sufficient titer.
Sanjuán, Moya, and Elena 2004 ). Briefly, young 90 per cent confluent BHK-21 cells were infected with a recombinant vaccinia virus expressing T7 RNA polymerase and then co-transfected with the full-length VSV cDNA clone and three helper plasmids encoding the P, L, and N proteins. Transfections were done using Lipofectamine LTX (Life Technologies), following manufacturer’s instructions. After 6 h, 25 µg/ml 1-β-D-arabinofuranosylcytosine was added to inhibit vaccinia replication. After 3–4 days, supernatants were tested for the presence of infectious VSV particles by the standard plaque assay, vaccinia virus was removed by filtration, and one additional VSV infection cycle was performed to reach sufficient titer.
8
Cloning and Mutagenesis of SOX9 and CEACAM1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
SOX9 expression vector was purchased from the plasmID Repository at Harvard Medical School (clone HsCD00004049). SOX9 cDNA was amplified from this vector and inserted into a pcDNA3 expression vector (Invitrogen, Carlsbad, CA, USA) using HindIII and XhoI restriction enzymes (New England Biolabs, Ipswich, MA, USA). DNA from melanoma cells for cloning of the CEACAM1 promoter was purified using GenElute Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich, St. Louis, MO, USA). Promoter fragments containing the full or partial putative promoter of CEACAM1 were amplified and cloned into pGL4.14 reporter vector (Promega, Madison, WI, USA) using XhoI and HindIII sites. Point mutations and deletions were introduced into the various constructs using specific primers, DNA synthesis with KOD Hot Start Polymerase (Merck Millipore, Darmstadt, Germany) and ultimately DpnI (New England Biolabs, Ipswich, MA, USA) digestion at 37°C for 1 hour. The full sequences of all primers used are detailed in Supplementary Table 1 .
9
Site-directed Mutagenesis of HIV Integrase
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The pPACKH1-GAG plasmid (SBI, Mountain View, CA) contains the structural (gag), replication (pol) and envelop (env) gene. The pol contains a functional domain of integrase, which is responsible for provirus integration. As shown in Fig. 1a , we designed and synthesized primer pairs to carry out site-directed mutagenesis using high fidelity DNA polymerase followed by DpnI (NEB, Ipswich, MA) treatment and transformation. The primers used for D64A (forward 5′- aggaatatggcaactagcttgtacacatttagaag-3′; reverse 5′-ctaaatgtgtacaagctagttgccatattcctggac), D116A (forward 5′- gtaaaaacaatacatacagccaatggcagcaatttcacc-3′; reverse 5′- gaaattgctgccattggctgtatgtattgtttttactggc-3′), D152A (forward 5′-caaagtcaaggagtagtagcatctatgaataaagaattaaagaaaatt; reverse 5′- ctttaattctttattcatagatgctactactccttgactttgggg-3′), and triplet mutation in basic region (forward5′-gtgacataaaagtagtgccagcagcacatgcaaagatcattagggat-3′; reverse primer 5′-atccctaatgatctttgcatgtgctgctggcactacttttatgtcac- 3′). Using different combinations of primer pairs, we have generated five integrase mutants as confirmed by double stranded DNA sequencing (SeqeneTech, Mountain View, California).
10
Construction and Characterization of Salmonella fliC Variants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The fliC gene and promoter elements were amplified from the Salmonella enterica serovar Typhimurium IR715 (“S. Typhimurium” throughout the text) chromosome using oligonucleotides fliC-F and fliC-R (Additional file 1 : Table S1) which encode restriction sites BamHI and EcoRI used for cloning into the pENTR3C vector (Invitrogen, USA), generating pFliC. Oligonucleotides encoding desired polymorphisms were used to divergently amplify the entire plasmid. Resulting PCR products were treated with DpnI (New England Biolabs, USA) to remove template plasmid prior to transformation into E. coli DH5α. Plasmid sequences were confirmed at the UC Riverside IIGB core facility. All pFliC variants were transformed into the S. Typhimurium SPN313 (IR715 ΔfliC fljB::MudCm) [7 (link)].
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!