phusion hot start dna polymerases  (Thermo Fisher)


Bioz Verified Symbol Thermo Fisher is a verified supplier  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    Phusion Hot Start II DNA Polymerase 2 U µL
    Description:
    Thermo Scientific Phusion High Fidelity DNA Polymerases set a gold standard for high performance PCR Featuring an error rate 50 fold lower than that of Taq and 6 fold lower than that of Pfu Phusion High Fidelity DNA Polymerase is excellent choice for cloning and other applications requiring high fidelity Phusion DNA Polymerases offer robust performance with short protocol times even in the presence of PCR inhibitors and generate higher yields with lower enzyme amounts than other DNA polymerase Using Phusion Hot Start II High Fidelity DNA Polymerase amplification proceeds without the production of nonspecific products due to the combination of Phusion DNA Polymerase and a reversibly bound specific Affibody ligand that inhibits DNA polymerase activity at room temperature The Affibody ligand also inhibits the 3 →5 exonuclease activity of the polymerase thus preventing degradation of primers and template DNA during reaction set up At temperatures that promote polymerase activity the ligand is released rendering the polymerase fully active Phusion Hot Start II DNA Polymerase is immediately reactivated at high temperatures so it does not require a separate activation step in PCR protocols Highlights• Reaction set up at room temperature• No non specific amplification and primer degradation during reaction set up• Zero time reactivation due to unique hot start technology• High fidelity 52X Taq • Fast PCR due to short extension times 15 30 s kb • Robust reactions minimal optimization needed• Increased product yields with minimal enzyme amounts• Available as a Green buffer format permitting direct loading on gels F 537S or F 537L Applications• High fidelity PCR• High throughput• Amplification of difficult GC rich templates• Template generation for sequencing• Multiplex PCR• Long range PCR• Cloning• Mutagenesis• MicroarrayUsing Phusion DNA Polymerases Annealing rules for Phusion DNA Polymerases are different from many common DNA polymerases such as Taq DNA polymerases For optimal results use our Tm calculator at www thermofisher com tmcalculator
    Catalog Number:
    f549l
    Price:
    None
    Applications:
    High Fidelity PCR|Hot Start PCR|PCR|PCR & Real-Time PCR
    Category:
    Proteins Enzymes Peptides
    Buy from Supplier


    Structured Review

    Thermo Fisher phusion hot start dna polymerases
    A diagram of the Hot Fusion process for single fragment cloning. Red and blue boxes on the vector and PCR product indicate the overlapping sequences (17–30 bp). T5 exonuclease removes nucleotides from the 5′ to 3′ end of double strand <t>DNA</t> molecules. <t>Phusion</t> DNA polymerase fills in gaps that are over-generated by T5 exonuclease. Annealed fragments are transformed into E. coli and the nick sites in the nucleotide chain are repaired by E. coli.
    Thermo Scientific Phusion High Fidelity DNA Polymerases set a gold standard for high performance PCR Featuring an error rate 50 fold lower than that of Taq and 6 fold lower than that of Pfu Phusion High Fidelity DNA Polymerase is excellent choice for cloning and other applications requiring high fidelity Phusion DNA Polymerases offer robust performance with short protocol times even in the presence of PCR inhibitors and generate higher yields with lower enzyme amounts than other DNA polymerase Using Phusion Hot Start II High Fidelity DNA Polymerase amplification proceeds without the production of nonspecific products due to the combination of Phusion DNA Polymerase and a reversibly bound specific Affibody ligand that inhibits DNA polymerase activity at room temperature The Affibody ligand also inhibits the 3 →5 exonuclease activity of the polymerase thus preventing degradation of primers and template DNA during reaction set up At temperatures that promote polymerase activity the ligand is released rendering the polymerase fully active Phusion Hot Start II DNA Polymerase is immediately reactivated at high temperatures so it does not require a separate activation step in PCR protocols Highlights• Reaction set up at room temperature• No non specific amplification and primer degradation during reaction set up• Zero time reactivation due to unique hot start technology• High fidelity 52X Taq • Fast PCR due to short extension times 15 30 s kb • Robust reactions minimal optimization needed• Increased product yields with minimal enzyme amounts• Available as a Green buffer format permitting direct loading on gels F 537S or F 537L Applications• High fidelity PCR• High throughput• Amplification of difficult GC rich templates• Template generation for sequencing• Multiplex PCR• Long range PCR• Cloning• Mutagenesis• MicroarrayUsing Phusion DNA Polymerases Annealing rules for Phusion DNA Polymerases are different from many common DNA polymerases such as Taq DNA polymerases For optimal results use our Tm calculator at www thermofisher com tmcalculator
    https://www.bioz.com/result/phusion hot start dna polymerases/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    phusion hot start dna polymerases - by Bioz Stars, 2020-04
    99/100 stars

    Related Products / Commonly Used Together

    generuler 1 kb plus dna ladder

    Images

    1) Product Images from "Hot Fusion: An Efficient Method to Clone Multiple DNA Fragments as Well as Inverted Repeats without Ligase"

    Article Title: Hot Fusion: An Efficient Method to Clone Multiple DNA Fragments as Well as Inverted Repeats without Ligase

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0115318

    A diagram of the Hot Fusion process for single fragment cloning. Red and blue boxes on the vector and PCR product indicate the overlapping sequences (17–30 bp). T5 exonuclease removes nucleotides from the 5′ to 3′ end of double strand DNA molecules. Phusion DNA polymerase fills in gaps that are over-generated by T5 exonuclease. Annealed fragments are transformed into E. coli and the nick sites in the nucleotide chain are repaired by E. coli.
    Figure Legend Snippet: A diagram of the Hot Fusion process for single fragment cloning. Red and blue boxes on the vector and PCR product indicate the overlapping sequences (17–30 bp). T5 exonuclease removes nucleotides from the 5′ to 3′ end of double strand DNA molecules. Phusion DNA polymerase fills in gaps that are over-generated by T5 exonuclease. Annealed fragments are transformed into E. coli and the nick sites in the nucleotide chain are repaired by E. coli.

    Techniques Used: Clone Assay, Plasmid Preparation, Polymerase Chain Reaction, Generated, Transformation Assay

    Related Articles

    Clone Assay:

    Article Title: Coregulated Genes Link Sulfide:Quinone Oxidoreductase and Arsenic Metabolism in Synechocystis sp. Strain PCC6803
    Article Snippet: Paragraph title: Cloning procedures and construction of mutant strains of Synechocystis . ... PCR amplification of the 3,832-bp DNA fragment containing sll5035, sll5036, and slr5037 coding sequences and slr5038 was performed using the 5038Reg2SalIFw and 5038Reg2NotRev primer pair ( ) using Synechocystis genomic DNA as the template and Phusion Hot Start polymerase (Finnzymes, Sweden) according to the manufacturer's recommendations.

    Article Title: Engineered baker’s yeast as whole-cell biocatalyst for one-pot stereo-selective conversion of amines to alcohols
    Article Snippet: Primers from MWG-Biotech AG (Ebersberg, Germany) and Phusion Hot Start II DNA Polymerase and dNTPs from Thermo Scientific (Rockford, IL, USA) were used for polymerase chain reactions (PCR) and performed in a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA, USA). .. InFusion® HD Cloning Kit (Clontech Laboratories, Mountain View, CA, USA) was used for DNA manipulation.

    Article Title: Effect of transcription terminator usage on the establishment of transgene transcriptional gene silencing
    Article Snippet: Generation of transformation plasmids GoldenBraid2.0 (GB) cloning system was used for cloning. .. Phusion Hot Start II DNA Polymerase (ThermoFisher) was used for DNA amplification.

    Article Title: CRISPR–Cas9-assisted recombineering in Lactobacillus reuteri
    Article Snippet: .. Polymerase chain reaction (PCR) amplifications for cloning purposes were performed with Phusion Hot Start II Polymerase (Fermentas), and PCR amplifications for screening purposes were performed with Taq DNA Polymerase (Denville Scientific). .. Pellet Paint Co-Precipitant (Novagen) was used to concentrate DNA for Gibson assembly or conventional T4 DNA ligase cloning.

    Amplification:

    Article Title: Coregulated Genes Link Sulfide:Quinone Oxidoreductase and Arsenic Metabolism in Synechocystis sp. Strain PCC6803
    Article Snippet: .. PCR amplification of the 3,832-bp DNA fragment containing sll5035, sll5036, and slr5037 coding sequences and slr5038 was performed using the 5038Reg2SalIFw and 5038Reg2NotRev primer pair ( ) using Synechocystis genomic DNA as the template and Phusion Hot Start polymerase (Finnzymes, Sweden) according to the manufacturer's recommendations. .. The PCR products were purified with the MinElute PCR purification kit (Qiagen, Germany).

    Article Title: Transcriptional networks specifying homeostatic and inflammatory programs of gene expression in human aortic endothelial cells
    Article Snippet: .. Libraries were then amplified by PCR (Phusion Hot Start II, ThermoFisher Scientific, #F549L) and purified (Zymo #D5205) for high-throughput sequencing. .. Chromatin immunoprecipitation sequencing (ChIP-seq) ChIP-seq was performed as previously described ( ).

    Article Title: The inner membrane histidine kinase EnvZ senses osmolality via helix-coil transitions in the cytoplasm
    Article Snippet: .. Site-specific mutation at H243 was introduced using 5′-ATG GCG GGG GTA AGT GCC GAC TTG CGC ACG CCG-3′ as the forward primer and 5′-CGG CGT GCG CAA GTC GGC ACT TAC CCC CGC CAT-3′ as the reverse primer and amplification was achieved using Phusion® Hot Start II DNA polymerase (Thermo Fisher Scientific, Lafayette, CO). .. Successful point mutation was determined using DNA sequencing and the mutant-containing plasmid was transformed into E. coli BL21 (DE3) competent cells.

    Article Title: Effect of transcription terminator usage on the establishment of transgene transcriptional gene silencing
    Article Snippet: .. Phusion Hot Start II DNA Polymerase (ThermoFisher) was used for DNA amplification. ..

    Article Title: An Extracellular Subtilase Switch for Immune Priming in Arabidopsis
    Article Snippet: .. For the SBT3.3m-GFP construct, pDONR207+SBT3.3 vector was amplified using Phusion Hot Start II polymerase (Thermo Scientific) with SBT3.3m FW and SBT3.3m RV phosphorylated primers including a T663 to G663 mutation. .. The PCR product was then digested with DpnI restriction enzyme (Fermentas), purified by Zymoclean DNA Recovery Kit (Zymo Research) and religated using T4 Ligase (Fermentas).

    Article Title: A Streamlined Approach to Antibody Novel Germline Allele Prediction and Validation
    Article Snippet: gDNA Sequencing and Reads Processing Nested PCR was used to reduce non-specific amplification. .. First PCR was performed on 10% of purified gDNA from 2,000 sorted T cells using Phusion Hot Start II DNA Polymerase (Thermo Scientific) with the following protocol: 98°C for 1 min; 10 cycles of 98°C for 30 s, 57°C for 1 min, and 72°C for 5 min; then 72°C for 10 min. Second PCR was performed on 10% of the first PCR product with the same protocol.

    Article Title: MAS promoter regulation: A role of Sry and tyrosine nitration of the KRAB domain of ZNF274 as a feedback mechanism
    Article Snippet: .. Mutations were incorporated into primers, vector amplified with those primers using Phusion Hot Start II (Thermo) and vectors ligated together using T4 ligase. .. GST pull down assays for Sry by the GST-KRAB-O construct were performed similarly to KRAB-KAP pull down experiments.

    Construct:

    Article Title: Coregulated Genes Link Sulfide:Quinone Oxidoreductase and Arsenic Metabolism in Synechocystis sp. Strain PCC6803
    Article Snippet: For various investigations, three deletion mutant strains, Synechocystis Δ suoRSCT , Δ suoR , and Δ suoT , were constructed by replacing the respective genomic regions with antibiotic resistance cassettes as follows. .. PCR amplification of the 3,832-bp DNA fragment containing sll5035, sll5036, and slr5037 coding sequences and slr5038 was performed using the 5038Reg2SalIFw and 5038Reg2NotRev primer pair ( ) using Synechocystis genomic DNA as the template and Phusion Hot Start polymerase (Finnzymes, Sweden) according to the manufacturer's recommendations.

    Article Title: An Extracellular Subtilase Switch for Immune Priming in Arabidopsis
    Article Snippet: .. For the SBT3.3m-GFP construct, pDONR207+SBT3.3 vector was amplified using Phusion Hot Start II polymerase (Thermo Scientific) with SBT3.3m FW and SBT3.3m RV phosphorylated primers including a T663 to G663 mutation. .. The PCR product was then digested with DpnI restriction enzyme (Fermentas), purified by Zymoclean DNA Recovery Kit (Zymo Research) and religated using T4 Ligase (Fermentas).

    Article Title: MAS promoter regulation: A role of Sry and tyrosine nitration of the KRAB domain of ZNF274 as a feedback mechanism
    Article Snippet: Mutations were incorporated into primers, vector amplified with those primers using Phusion Hot Start II (Thermo) and vectors ligated together using T4 ligase. .. GST pull down assays for Sry by the GST-KRAB-O construct were performed similarly to KRAB-KAP pull down experiments.

    Incubation:

    Article Title: MAS promoter regulation: A role of Sry and tyrosine nitration of the KRAB domain of ZNF274 as a feedback mechanism
    Article Snippet: Membranes were washed four times with PBS-T, incubated in anti-Rabbit antibody congregated with HRP in blocking buffer for one hour at room temperature, washed four times with PBS-T, and imaged with SuperSignal West Pico Chemiluminescent substrate (Thermo-Fisher) on X-ray film. .. Mutations were incorporated into primers, vector amplified with those primers using Phusion Hot Start II (Thermo) and vectors ligated together using T4 ligase.

    Luciferase:

    Article Title: Effect of transcription terminator usage on the establishment of transgene transcriptional gene silencing
    Article Snippet: Firefly luciferase was amplified from GB0255 using primers 1678 (5′-GCGCCGTCTCGCTCGAATGGAAGACGCCAAAAACATAAAG-3′)/1679 (5′-GCGCCGTCTCGCTCGCTGCTTACACGGCGATCTTTCCGC-3′). .. Phusion Hot Start II DNA Polymerase (ThermoFisher) was used for DNA amplification.

    Expressing:

    Article Title: Highly Expandable Human iPS Cell-Derived Neural Progenitor Cells (NPC) and Neurons for Central Nervous System Disease Modeling and High-Throughput Screening
    Article Snippet: .. pTetO-mNgn2-TA-puro ( ; available at Addgene plasmid ID# 52047) pLX-304 (Addgene plasmid ID# 25890) Phusion Hot Start II DNA polymerase (ThermoFisher Scientific cat# F549L) PacI restriction endonuclease (NEB cat# R0547S) Calf intestinal alkaline phosphatase (NEB cat# M0290S) Quick Ligation kit (NEB cat# M2200S) Stbl3 E. coli strain (ThermoFisher Scientific cat# C737303) Qiagen Plasmid Maxi Kit (cat# 12162) Transactivator plasmid pFUW-M2rtTA ( , Addgene plasmid ID# 20342 Lentiviral packaging plasmid pCMV-dR8.2 dvpr ( , Addgene plasmid ID# 8455 VSV-G envelope expressing plasmid pMD2.G (Addgene plasmid ID# 12259) Humidified incubator at 37°C with 5% CO2 HEK293T cells (ATCC 293T/17) Poly-L-Lysine (Sigma cat# P5899) D10 medium (see recipe) Lipofectamine 2000 (Thermo Fisher Scientific cat# 11668-019) Opti-MEM (Gibco cat# 31985) Neural proliferation media (NPM, see recipe) POL-coated plates (see recipe) Blasticidin (Gibco cat# A11139-03) POLS-coated plates iNgn2 neural media (N3aM, see recipe) Puromycin (Sigma cat# P8833-25MG) Cytosine arabinoside (AraC; Sigma cat# C6645) .. There are numerous selection markers and many methods to insert the selection marker of choice into the inducible Ngn2 construct (pTetO-mNgn2-TA-puro).

    Modification:

    Article Title: CRISPR–Cas9-assisted recombineering in Lactobacillus reuteri
    Article Snippet: Reagents and enzymes All modification enzymes were purchased from Fermentas. .. Polymerase chain reaction (PCR) amplifications for cloning purposes were performed with Phusion Hot Start II Polymerase (Fermentas), and PCR amplifications for screening purposes were performed with Taq DNA Polymerase (Denville Scientific).

    Western Blot:

    Article Title: MAS promoter regulation: A role of Sry and tyrosine nitration of the KRAB domain of ZNF274 as a feedback mechanism
    Article Snippet: Mutations were incorporated into primers, vector amplified with those primers using Phusion Hot Start II (Thermo) and vectors ligated together using T4 ligase. .. Western blot for the His tag was performed by transferring the gel to a nitrocellulose membrane, blocking in 5% milk, probing with the His-probe (G-18, Santa Cruz Biotechnology), washed in PBS-T, treated with secondary donkey anti-Goat HRP antibody, washed in PBS-T, and chemiluminescence measured on film.

    Transformation Assay:

    Article Title: The inner membrane histidine kinase EnvZ senses osmolality via helix-coil transitions in the cytoplasm
    Article Snippet: Site-specific mutation at H243 was introduced using 5′-ATG GCG GGG GTA AGT GCC GAC TTG CGC ACG CCG-3′ as the forward primer and 5′-CGG CGT GCG CAA GTC GGC ACT TAC CCC CGC CAT-3′ as the reverse primer and amplification was achieved using Phusion® Hot Start II DNA polymerase (Thermo Fisher Scientific, Lafayette, CO). .. Successful point mutation was determined using DNA sequencing and the mutant-containing plasmid was transformed into E. coli BL21 (DE3) competent cells.

    Article Title: Effect of transcription terminator usage on the establishment of transgene transcriptional gene silencing
    Article Snippet: Paragraph title: Generation of transformation plasmids ... Phusion Hot Start II DNA Polymerase (ThermoFisher) was used for DNA amplification.

    Article Title: An Extracellular Subtilase Switch for Immune Priming in Arabidopsis
    Article Snippet: For the SBT3.3m-GFP construct, pDONR207+SBT3.3 vector was amplified using Phusion Hot Start II polymerase (Thermo Scientific) with SBT3.3m FW and SBT3.3m RV phosphorylated primers including a T663 to G663 mutation. .. After sequencing, all constructs were recombined with pB7FWG destination vector using LR ClonaseMixII kit (Invitrogen) and introduced into Arabidopsis (Col-0) via Agrobacterium transformation.

    Over Expression:

    Article Title: The inner membrane histidine kinase EnvZ senses osmolality via helix-coil transitions in the cytoplasm
    Article Snippet: Site-specific mutation at H243 was introduced using 5′-ATG GCG GGG GTA AGT GCC GAC TTG CGC ACG CCG-3′ as the forward primer and 5′-CGG CGT GCG CAA GTC GGC ACT TAC CCC CGC CAT-3′ as the reverse primer and amplification was achieved using Phusion® Hot Start II DNA polymerase (Thermo Fisher Scientific, Lafayette, CO). .. Overexpression and purification of H243A EnvZc protein was as described above for wild-type EnvZc .

    Activated Clotting Time Assay:

    Article Title: The inner membrane histidine kinase EnvZ senses osmolality via helix-coil transitions in the cytoplasm
    Article Snippet: .. Site-specific mutation at H243 was introduced using 5′-ATG GCG GGG GTA AGT GCC GAC TTG CGC ACG CCG-3′ as the forward primer and 5′-CGG CGT GCG CAA GTC GGC ACT TAC CCC CGC CAT-3′ as the reverse primer and amplification was achieved using Phusion® Hot Start II DNA polymerase (Thermo Fisher Scientific, Lafayette, CO). .. Successful point mutation was determined using DNA sequencing and the mutant-containing plasmid was transformed into E. coli BL21 (DE3) competent cells.

    Countercurrent Chromatography:

    Article Title: The inner membrane histidine kinase EnvZ senses osmolality via helix-coil transitions in the cytoplasm
    Article Snippet: .. Site-specific mutation at H243 was introduced using 5′-ATG GCG GGG GTA AGT GCC GAC TTG CGC ACG CCG-3′ as the forward primer and 5′-CGG CGT GCG CAA GTC GGC ACT TAC CCC CGC CAT-3′ as the reverse primer and amplification was achieved using Phusion® Hot Start II DNA polymerase (Thermo Fisher Scientific, Lafayette, CO). .. Successful point mutation was determined using DNA sequencing and the mutant-containing plasmid was transformed into E. coli BL21 (DE3) competent cells.

    Inverse PCR:

    Article Title: The inner membrane histidine kinase EnvZ senses osmolality via helix-coil transitions in the cytoplasm
    Article Snippet: The H243A mutant of EnvZc was generated using inverse polymerase chain reaction (PCR) method and pET11a- envZ c as the template. .. Site-specific mutation at H243 was introduced using 5′-ATG GCG GGG GTA AGT GCC GAC TTG CGC ACG CCG-3′ as the forward primer and 5′-CGG CGT GCG CAA GTC GGC ACT TAC CCC CGC CAT-3′ as the reverse primer and amplification was achieved using Phusion® Hot Start II DNA polymerase (Thermo Fisher Scientific, Lafayette, CO).

    Nitration:

    Article Title: MAS promoter regulation: A role of Sry and tyrosine nitration of the KRAB domain of ZNF274 as a feedback mechanism
    Article Snippet: Paragraph title: Tyrosine nitration of ZNF274 and KAP-1 proteins ... Mutations were incorporated into primers, vector amplified with those primers using Phusion Hot Start II (Thermo) and vectors ligated together using T4 ligase.

    Ligation:

    Article Title: A Streamlined Approach to Antibody Novel Germline Allele Prediction and Validation
    Article Snippet: First PCR was performed on 10% of purified gDNA from 2,000 sorted T cells using Phusion Hot Start II DNA Polymerase (Thermo Scientific) with the following protocol: 98°C for 1 min; 10 cycles of 98°C for 30 s, 57°C for 1 min, and 72°C for 5 min; then 72°C for 10 min. Second PCR was performed on 10% of the first PCR product with the same protocol. .. Final adaptor ligation was performed on 10% of the second PCR product using TaKaRa Ex Taq DNA Polymerase Hot Start with the following protocol: 95°C for 3 min; 10 cycles of 95°C for 30 s, 57°C for 30 s, and 72°C for 2 min; then 72°C for 7 min.

    Article Title: Highly Expandable Human iPS Cell-Derived Neural Progenitor Cells (NPC) and Neurons for Central Nervous System Disease Modeling and High-Throughput Screening
    Article Snippet: .. pTetO-mNgn2-TA-puro ( ; available at Addgene plasmid ID# 52047) pLX-304 (Addgene plasmid ID# 25890) Phusion Hot Start II DNA polymerase (ThermoFisher Scientific cat# F549L) PacI restriction endonuclease (NEB cat# R0547S) Calf intestinal alkaline phosphatase (NEB cat# M0290S) Quick Ligation kit (NEB cat# M2200S) Stbl3 E. coli strain (ThermoFisher Scientific cat# C737303) Qiagen Plasmid Maxi Kit (cat# 12162) Transactivator plasmid pFUW-M2rtTA ( , Addgene plasmid ID# 20342 Lentiviral packaging plasmid pCMV-dR8.2 dvpr ( , Addgene plasmid ID# 8455 VSV-G envelope expressing plasmid pMD2.G (Addgene plasmid ID# 12259) Humidified incubator at 37°C with 5% CO2 HEK293T cells (ATCC 293T/17) Poly-L-Lysine (Sigma cat# P5899) D10 medium (see recipe) Lipofectamine 2000 (Thermo Fisher Scientific cat# 11668-019) Opti-MEM (Gibco cat# 31985) Neural proliferation media (NPM, see recipe) POL-coated plates (see recipe) Blasticidin (Gibco cat# A11139-03) POLS-coated plates iNgn2 neural media (N3aM, see recipe) Puromycin (Sigma cat# P8833-25MG) Cytosine arabinoside (AraC; Sigma cat# C6645) .. There are numerous selection markers and many methods to insert the selection marker of choice into the inducible Ngn2 construct (pTetO-mNgn2-TA-puro).

    Atomic Absorption Spectroscopy:

    Article Title: Effect of transcription terminator usage on the establishment of transgene transcriptional gene silencing
    Article Snippet: FUS3short was amplified from Arabidopsis genomic DNA using primers 1668 (5′-GCGCCGTCTCGCTCGGCAGGGAAATGTTCTTACTATTATCCAGTCAT-3′)/1676 (5′-GCGCCGTCTCGCTCGAAGCTTATCCACCCAAAAAATCGAG-3′) to include FUS3 AAs 246–283. .. Phusion Hot Start II DNA Polymerase (ThermoFisher) was used for DNA amplification.

    Transferring:

    Article Title: MAS promoter regulation: A role of Sry and tyrosine nitration of the KRAB domain of ZNF274 as a feedback mechanism
    Article Snippet: Mutations were incorporated into primers, vector amplified with those primers using Phusion Hot Start II (Thermo) and vectors ligated together using T4 ligase. .. Western blot for the His tag was performed by transferring the gel to a nitrocellulose membrane, blocking in 5% milk, probing with the His-probe (G-18, Santa Cruz Biotechnology), washed in PBS-T, treated with secondary donkey anti-Goat HRP antibody, washed in PBS-T, and chemiluminescence measured on film.

    Generated:

    Article Title: The inner membrane histidine kinase EnvZ senses osmolality via helix-coil transitions in the cytoplasm
    Article Snippet: The H243A mutant of EnvZc was generated using inverse polymerase chain reaction (PCR) method and pET11a- envZ c as the template. .. Site-specific mutation at H243 was introduced using 5′-ATG GCG GGG GTA AGT GCC GAC TTG CGC ACG CCG-3′ as the forward primer and 5′-CGG CGT GCG CAA GTC GGC ACT TAC CCC CGC CAT-3′ as the reverse primer and amplification was achieved using Phusion® Hot Start II DNA polymerase (Thermo Fisher Scientific, Lafayette, CO).

    DNA Sequencing:

    Article Title: The inner membrane histidine kinase EnvZ senses osmolality via helix-coil transitions in the cytoplasm
    Article Snippet: Site-specific mutation at H243 was introduced using 5′-ATG GCG GGG GTA AGT GCC GAC TTG CGC ACG CCG-3′ as the forward primer and 5′-CGG CGT GCG CAA GTC GGC ACT TAC CCC CGC CAT-3′ as the reverse primer and amplification was achieved using Phusion® Hot Start II DNA polymerase (Thermo Fisher Scientific, Lafayette, CO). .. Successful point mutation was determined using DNA sequencing and the mutant-containing plasmid was transformed into E. coli BL21 (DE3) competent cells.

    Sequencing:

    Article Title: Transcriptional networks specifying homeostatic and inflammatory programs of gene expression in human aortic endothelial cells
    Article Snippet: .. Libraries were then amplified by PCR (Phusion Hot Start II, ThermoFisher Scientific, #F549L) and purified (Zymo #D5205) for high-throughput sequencing. .. Chromatin immunoprecipitation sequencing (ChIP-seq) ChIP-seq was performed as previously described ( ).

    Article Title: An Extracellular Subtilase Switch for Immune Priming in Arabidopsis
    Article Snippet: For the SBT3.3m-GFP construct, pDONR207+SBT3.3 vector was amplified using Phusion Hot Start II polymerase (Thermo Scientific) with SBT3.3m FW and SBT3.3m RV phosphorylated primers including a T663 to G663 mutation. .. After sequencing, all constructs were recombined with pB7FWG destination vector using LR ClonaseMixII kit (Invitrogen) and introduced into Arabidopsis (Col-0) via Agrobacterium transformation.

    Article Title: A Streamlined Approach to Antibody Novel Germline Allele Prediction and Validation
    Article Snippet: Paragraph title: gDNA Sequencing and Reads Processing ... First PCR was performed on 10% of purified gDNA from 2,000 sorted T cells using Phusion Hot Start II DNA Polymerase (Thermo Scientific) with the following protocol: 98°C for 1 min; 10 cycles of 98°C for 30 s, 57°C for 1 min, and 72°C for 5 min; then 72°C for 10 min. Second PCR was performed on 10% of the first PCR product with the same protocol.

    Binding Assay:

    Article Title: MAS promoter regulation: A role of Sry and tyrosine nitration of the KRAB domain of ZNF274 as a feedback mechanism
    Article Snippet: Amino acids in Sry critical to binding KRAB were determined by site directed mutagenesis of the pET28 full length human Sry (expressed and purified as above). .. Mutations were incorporated into primers, vector amplified with those primers using Phusion Hot Start II (Thermo) and vectors ligated together using T4 ligase.

    Cellular Antioxidant Activity Assay:

    Article Title: The inner membrane histidine kinase EnvZ senses osmolality via helix-coil transitions in the cytoplasm
    Article Snippet: .. Site-specific mutation at H243 was introduced using 5′-ATG GCG GGG GTA AGT GCC GAC TTG CGC ACG CCG-3′ as the forward primer and 5′-CGG CGT GCG CAA GTC GGC ACT TAC CCC CGC CAT-3′ as the reverse primer and amplification was achieved using Phusion® Hot Start II DNA polymerase (Thermo Fisher Scientific, Lafayette, CO). .. Successful point mutation was determined using DNA sequencing and the mutant-containing plasmid was transformed into E. coli BL21 (DE3) competent cells.

    DNA Extraction:

    Article Title: Engineered baker’s yeast as whole-cell biocatalyst for one-pot stereo-selective conversion of amines to alcohols
    Article Snippet: Nucleic acid manipulation Plasmid DNA was prepared with the GeneJET Plasmid Miniprep Kit (Thermo Scientific, Rockford, IL, USA) and agarose gel DNA extraction was performed using QIAquick® Gel Extraction Kit (Qiagen GmbH, Hilden, Germany). .. Primers from MWG-Biotech AG (Ebersberg, Germany) and Phusion Hot Start II DNA Polymerase and dNTPs from Thermo Scientific (Rockford, IL, USA) were used for polymerase chain reactions (PCR) and performed in a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA, USA).

    RNA Sequencing Assay:

    Article Title: Transcriptional networks specifying homeostatic and inflammatory programs of gene expression in human aortic endothelial cells
    Article Snippet: Paragraph title: RNA-seq ... Libraries were then amplified by PCR (Phusion Hot Start II, ThermoFisher Scientific, #F549L) and purified (Zymo #D5205) for high-throughput sequencing.

    Mutagenesis:

    Article Title: Coregulated Genes Link Sulfide:Quinone Oxidoreductase and Arsenic Metabolism in Synechocystis sp. Strain PCC6803
    Article Snippet: Paragraph title: Cloning procedures and construction of mutant strains of Synechocystis . ... PCR amplification of the 3,832-bp DNA fragment containing sll5035, sll5036, and slr5037 coding sequences and slr5038 was performed using the 5038Reg2SalIFw and 5038Reg2NotRev primer pair ( ) using Synechocystis genomic DNA as the template and Phusion Hot Start polymerase (Finnzymes, Sweden) according to the manufacturer's recommendations.

    Article Title: The inner membrane histidine kinase EnvZ senses osmolality via helix-coil transitions in the cytoplasm
    Article Snippet: .. Site-specific mutation at H243 was introduced using 5′-ATG GCG GGG GTA AGT GCC GAC TTG CGC ACG CCG-3′ as the forward primer and 5′-CGG CGT GCG CAA GTC GGC ACT TAC CCC CGC CAT-3′ as the reverse primer and amplification was achieved using Phusion® Hot Start II DNA polymerase (Thermo Fisher Scientific, Lafayette, CO). .. Successful point mutation was determined using DNA sequencing and the mutant-containing plasmid was transformed into E. coli BL21 (DE3) competent cells.

    Article Title: An Extracellular Subtilase Switch for Immune Priming in Arabidopsis
    Article Snippet: .. For the SBT3.3m-GFP construct, pDONR207+SBT3.3 vector was amplified using Phusion Hot Start II polymerase (Thermo Scientific) with SBT3.3m FW and SBT3.3m RV phosphorylated primers including a T663 to G663 mutation. .. The PCR product was then digested with DpnI restriction enzyme (Fermentas), purified by Zymoclean DNA Recovery Kit (Zymo Research) and religated using T4 Ligase (Fermentas).

    Article Title: MAS promoter regulation: A role of Sry and tyrosine nitration of the KRAB domain of ZNF274 as a feedback mechanism
    Article Snippet: Amino acids in Sry critical to binding KRAB were determined by site directed mutagenesis of the pET28 full length human Sry (expressed and purified as above). .. Mutations were incorporated into primers, vector amplified with those primers using Phusion Hot Start II (Thermo) and vectors ligated together using T4 ligase.

    Isolation:

    Article Title: Transcriptional networks specifying homeostatic and inflammatory programs of gene expression in human aortic endothelial cells
    Article Snippet: RNA-seq HAECs were resuspended in RNA Lysis Buffer and RNA was extracted from cells using the Quick-RNA Micro Prep kit from ZymoResearch (Irvine, CA, #R1051), including optional DNase I treatment. mRNA was selected through poly-A isolation using Oligo d(T)25 beads (New England BioLabs, Ipswich, MA, #S1419S). .. Libraries were then amplified by PCR (Phusion Hot Start II, ThermoFisher Scientific, #F549L) and purified (Zymo #D5205) for high-throughput sequencing.

    Purification:

    Article Title: Coregulated Genes Link Sulfide:Quinone Oxidoreductase and Arsenic Metabolism in Synechocystis sp. Strain PCC6803
    Article Snippet: PCR amplification of the 3,832-bp DNA fragment containing sll5035, sll5036, and slr5037 coding sequences and slr5038 was performed using the 5038Reg2SalIFw and 5038Reg2NotRev primer pair ( ) using Synechocystis genomic DNA as the template and Phusion Hot Start polymerase (Finnzymes, Sweden) according to the manufacturer's recommendations. .. The PCR products were purified with the MinElute PCR purification kit (Qiagen, Germany).

    Article Title: Hot Fusion: An Efficient Method to Clone Multiple DNA Fragments as Well as Inverted Repeats without Ligase
    Article Snippet: Phusion Hot Start DNA polymerases and GeneRuler 1 kb Plus DNA Ladder were purchased from Thermo Scientific (Waltham, MA). .. DNA and PCR product purification kits were purchased from Qiagen (Germany) and Zymo Research Corp (Orange, CA).

    Article Title: Transcriptional networks specifying homeostatic and inflammatory programs of gene expression in human aortic endothelial cells
    Article Snippet: .. Libraries were then amplified by PCR (Phusion Hot Start II, ThermoFisher Scientific, #F549L) and purified (Zymo #D5205) for high-throughput sequencing. .. Chromatin immunoprecipitation sequencing (ChIP-seq) ChIP-seq was performed as previously described ( ).

    Article Title: Engineered baker’s yeast as whole-cell biocatalyst for one-pot stereo-selective conversion of amines to alcohols
    Article Snippet: Primers from MWG-Biotech AG (Ebersberg, Germany) and Phusion Hot Start II DNA Polymerase and dNTPs from Thermo Scientific (Rockford, IL, USA) were used for polymerase chain reactions (PCR) and performed in a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA, USA). .. PCR products were purified with the GeneJET PCR Purification Kit (Thermo Scientific, Rockford, IL, USA).

    Article Title: The inner membrane histidine kinase EnvZ senses osmolality via helix-coil transitions in the cytoplasm
    Article Snippet: Site-specific mutation at H243 was introduced using 5′-ATG GCG GGG GTA AGT GCC GAC TTG CGC ACG CCG-3′ as the forward primer and 5′-CGG CGT GCG CAA GTC GGC ACT TAC CCC CGC CAT-3′ as the reverse primer and amplification was achieved using Phusion® Hot Start II DNA polymerase (Thermo Fisher Scientific, Lafayette, CO). .. Overexpression and purification of H243A EnvZc protein was as described above for wild-type EnvZc .

    Article Title: An Extracellular Subtilase Switch for Immune Priming in Arabidopsis
    Article Snippet: For the SBT3.3m-GFP construct, pDONR207+SBT3.3 vector was amplified using Phusion Hot Start II polymerase (Thermo Scientific) with SBT3.3m FW and SBT3.3m RV phosphorylated primers including a T663 to G663 mutation. .. The PCR product was then digested with DpnI restriction enzyme (Fermentas), purified by Zymoclean DNA Recovery Kit (Zymo Research) and religated using T4 Ligase (Fermentas).

    Article Title: A Streamlined Approach to Antibody Novel Germline Allele Prediction and Validation
    Article Snippet: .. First PCR was performed on 10% of purified gDNA from 2,000 sorted T cells using Phusion Hot Start II DNA Polymerase (Thermo Scientific) with the following protocol: 98°C for 1 min; 10 cycles of 98°C for 30 s, 57°C for 1 min, and 72°C for 5 min; then 72°C for 10 min. Second PCR was performed on 10% of the first PCR product with the same protocol. .. Final adaptor ligation was performed on 10% of the second PCR product using TaKaRa Ex Taq DNA Polymerase Hot Start with the following protocol: 95°C for 3 min; 10 cycles of 95°C for 30 s, 57°C for 30 s, and 72°C for 2 min; then 72°C for 7 min.

    Article Title: MAS promoter regulation: A role of Sry and tyrosine nitration of the KRAB domain of ZNF274 as a feedback mechanism
    Article Snippet: Amino acids in Sry critical to binding KRAB were determined by site directed mutagenesis of the pET28 full length human Sry (expressed and purified as above). .. Mutations were incorporated into primers, vector amplified with those primers using Phusion Hot Start II (Thermo) and vectors ligated together using T4 ligase.

    Polymerase Chain Reaction:

    Article Title: Coregulated Genes Link Sulfide:Quinone Oxidoreductase and Arsenic Metabolism in Synechocystis sp. Strain PCC6803
    Article Snippet: .. PCR amplification of the 3,832-bp DNA fragment containing sll5035, sll5036, and slr5037 coding sequences and slr5038 was performed using the 5038Reg2SalIFw and 5038Reg2NotRev primer pair ( ) using Synechocystis genomic DNA as the template and Phusion Hot Start polymerase (Finnzymes, Sweden) according to the manufacturer's recommendations. .. The PCR products were purified with the MinElute PCR purification kit (Qiagen, Germany).

    Article Title: Hot Fusion: An Efficient Method to Clone Multiple DNA Fragments as Well as Inverted Repeats without Ligase
    Article Snippet: Phusion Hot Start DNA polymerases and GeneRuler 1 kb Plus DNA Ladder were purchased from Thermo Scientific (Waltham, MA). .. DNA and PCR product purification kits were purchased from Qiagen (Germany) and Zymo Research Corp (Orange, CA).

    Article Title: Sequencing Antibody Repertoires Provides Evidence for Original Antigenic Sin Shaping the Antibody Response to Influenza Vaccination
    Article Snippet: .. We used Phusion Hot Start II DNA polymerase (NEB/Fermentas) for both the first PCR (PCR1) and the nested PCR (PCR2). ..

    Article Title: Transcriptional networks specifying homeostatic and inflammatory programs of gene expression in human aortic endothelial cells
    Article Snippet: .. Libraries were then amplified by PCR (Phusion Hot Start II, ThermoFisher Scientific, #F549L) and purified (Zymo #D5205) for high-throughput sequencing. .. Chromatin immunoprecipitation sequencing (ChIP-seq) ChIP-seq was performed as previously described ( ).

    Article Title: Engineered baker’s yeast as whole-cell biocatalyst for one-pot stereo-selective conversion of amines to alcohols
    Article Snippet: .. Primers from MWG-Biotech AG (Ebersberg, Germany) and Phusion Hot Start II DNA Polymerase and dNTPs from Thermo Scientific (Rockford, IL, USA) were used for polymerase chain reactions (PCR) and performed in a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA, USA). .. PCR products were purified with the GeneJET PCR Purification Kit (Thermo Scientific, Rockford, IL, USA).

    Article Title: The inner membrane histidine kinase EnvZ senses osmolality via helix-coil transitions in the cytoplasm
    Article Snippet: The H243A mutant of EnvZc was generated using inverse polymerase chain reaction (PCR) method and pET11a- envZ c as the template. .. Site-specific mutation at H243 was introduced using 5′-ATG GCG GGG GTA AGT GCC GAC TTG CGC ACG CCG-3′ as the forward primer and 5′-CGG CGT GCG CAA GTC GGC ACT TAC CCC CGC CAT-3′ as the reverse primer and amplification was achieved using Phusion® Hot Start II DNA polymerase (Thermo Fisher Scientific, Lafayette, CO).

    Article Title: CRISPR–Cas9-assisted recombineering in Lactobacillus reuteri
    Article Snippet: .. Polymerase chain reaction (PCR) amplifications for cloning purposes were performed with Phusion Hot Start II Polymerase (Fermentas), and PCR amplifications for screening purposes were performed with Taq DNA Polymerase (Denville Scientific). .. Pellet Paint Co-Precipitant (Novagen) was used to concentrate DNA for Gibson assembly or conventional T4 DNA ligase cloning.

    Article Title: An Extracellular Subtilase Switch for Immune Priming in Arabidopsis
    Article Snippet: Gene constructs and transgenic lines For the SBT3.3-GFP overexpressing construct, a full length cDNA for SBT3.3 was amplified by PCR using Pfu DNA polymerase (Stratagene, San Diego, CA) and specific primers including Gateway adapters: BP SBT3.3 FW and BP SBT3.3 RV and recombined into pDONR207 using BP ClonaseMixII kit (Invitrogen). .. For the SBT3.3m-GFP construct, pDONR207+SBT3.3 vector was amplified using Phusion Hot Start II polymerase (Thermo Scientific) with SBT3.3m FW and SBT3.3m RV phosphorylated primers including a T663 to G663 mutation.

    Article Title: A Streamlined Approach to Antibody Novel Germline Allele Prediction and Validation
    Article Snippet: .. First PCR was performed on 10% of purified gDNA from 2,000 sorted T cells using Phusion Hot Start II DNA Polymerase (Thermo Scientific) with the following protocol: 98°C for 1 min; 10 cycles of 98°C for 30 s, 57°C for 1 min, and 72°C for 5 min; then 72°C for 10 min. Second PCR was performed on 10% of the first PCR product with the same protocol. .. Final adaptor ligation was performed on 10% of the second PCR product using TaKaRa Ex Taq DNA Polymerase Hot Start with the following protocol: 95°C for 3 min; 10 cycles of 95°C for 30 s, 57°C for 30 s, and 72°C for 2 min; then 72°C for 7 min.

    Blocking Assay:

    Article Title: MAS promoter regulation: A role of Sry and tyrosine nitration of the KRAB domain of ZNF274 as a feedback mechanism
    Article Snippet: Membranes were washed four times with PBS-T, incubated in anti-Rabbit antibody congregated with HRP in blocking buffer for one hour at room temperature, washed four times with PBS-T, and imaged with SuperSignal West Pico Chemiluminescent substrate (Thermo-Fisher) on X-ray film. .. Mutations were incorporated into primers, vector amplified with those primers using Phusion Hot Start II (Thermo) and vectors ligated together using T4 ligase.

    Gel Extraction:

    Article Title: Engineered baker’s yeast as whole-cell biocatalyst for one-pot stereo-selective conversion of amines to alcohols
    Article Snippet: Nucleic acid manipulation Plasmid DNA was prepared with the GeneJET Plasmid Miniprep Kit (Thermo Scientific, Rockford, IL, USA) and agarose gel DNA extraction was performed using QIAquick® Gel Extraction Kit (Qiagen GmbH, Hilden, Germany). .. Primers from MWG-Biotech AG (Ebersberg, Germany) and Phusion Hot Start II DNA Polymerase and dNTPs from Thermo Scientific (Rockford, IL, USA) were used for polymerase chain reactions (PCR) and performed in a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA, USA).

    Nested PCR:

    Article Title: Sequencing Antibody Repertoires Provides Evidence for Original Antigenic Sin Shaping the Antibody Response to Influenza Vaccination
    Article Snippet: .. We used Phusion Hot Start II DNA polymerase (NEB/Fermentas) for both the first PCR (PCR1) and the nested PCR (PCR2). ..

    Article Title: A Streamlined Approach to Antibody Novel Germline Allele Prediction and Validation
    Article Snippet: gDNA Sequencing and Reads Processing Nested PCR was used to reduce non-specific amplification. .. First PCR was performed on 10% of purified gDNA from 2,000 sorted T cells using Phusion Hot Start II DNA Polymerase (Thermo Scientific) with the following protocol: 98°C for 1 min; 10 cycles of 98°C for 30 s, 57°C for 1 min, and 72°C for 5 min; then 72°C for 10 min. Second PCR was performed on 10% of the first PCR product with the same protocol.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: The inner membrane histidine kinase EnvZ senses osmolality via helix-coil transitions in the cytoplasm
    Article Snippet: .. Site-specific mutation at H243 was introduced using 5′-ATG GCG GGG GTA AGT GCC GAC TTG CGC ACG CCG-3′ as the forward primer and 5′-CGG CGT GCG CAA GTC GGC ACT TAC CCC CGC CAT-3′ as the reverse primer and amplification was achieved using Phusion® Hot Start II DNA polymerase (Thermo Fisher Scientific, Lafayette, CO). .. Successful point mutation was determined using DNA sequencing and the mutant-containing plasmid was transformed into E. coli BL21 (DE3) competent cells.

    Plasmid Preparation:

    Article Title: Coregulated Genes Link Sulfide:Quinone Oxidoreductase and Arsenic Metabolism in Synechocystis sp. Strain PCC6803
    Article Snippet: For all cloning purposes, the pBluescript II SK+ (pBluescript) vector was used. .. PCR amplification of the 3,832-bp DNA fragment containing sll5035, sll5036, and slr5037 coding sequences and slr5038 was performed using the 5038Reg2SalIFw and 5038Reg2NotRev primer pair ( ) using Synechocystis genomic DNA as the template and Phusion Hot Start polymerase (Finnzymes, Sweden) according to the manufacturer's recommendations.

    Article Title: Hot Fusion: An Efficient Method to Clone Multiple DNA Fragments as Well as Inverted Repeats without Ligase
    Article Snippet: Phusion Hot Start DNA polymerases and GeneRuler 1 kb Plus DNA Ladder were purchased from Thermo Scientific (Waltham, MA). .. Most chemicals and antibiotics were purchased from Sigma-Aldrich (St. Louis, MO). pSV-β-Gal vector containing lacZ was purchased from Promega (Madison, WI).

    Article Title: Engineered baker’s yeast as whole-cell biocatalyst for one-pot stereo-selective conversion of amines to alcohols
    Article Snippet: Nucleic acid manipulation Plasmid DNA was prepared with the GeneJET Plasmid Miniprep Kit (Thermo Scientific, Rockford, IL, USA) and agarose gel DNA extraction was performed using QIAquick® Gel Extraction Kit (Qiagen GmbH, Hilden, Germany). .. Primers from MWG-Biotech AG (Ebersberg, Germany) and Phusion Hot Start II DNA Polymerase and dNTPs from Thermo Scientific (Rockford, IL, USA) were used for polymerase chain reactions (PCR) and performed in a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA, USA).

    Article Title: The inner membrane histidine kinase EnvZ senses osmolality via helix-coil transitions in the cytoplasm
    Article Snippet: Site-specific mutation at H243 was introduced using 5′-ATG GCG GGG GTA AGT GCC GAC TTG CGC ACG CCG-3′ as the forward primer and 5′-CGG CGT GCG CAA GTC GGC ACT TAC CCC CGC CAT-3′ as the reverse primer and amplification was achieved using Phusion® Hot Start II DNA polymerase (Thermo Fisher Scientific, Lafayette, CO). .. Successful point mutation was determined using DNA sequencing and the mutant-containing plasmid was transformed into E. coli BL21 (DE3) competent cells.

    Article Title: Effect of transcription terminator usage on the establishment of transgene transcriptional gene silencing
    Article Snippet: Phusion Hot Start II DNA Polymerase (ThermoFisher) was used for DNA amplification. .. These three constructions were assembled with GB0235 into pDGB_2omega1, and again assembled with GB0466, a “twister” plasmid that contains a 150 bp stuffer fragment in order to get the constructions into pDBG_2alpha1 as the final plasmid.

    Article Title: An Extracellular Subtilase Switch for Immune Priming in Arabidopsis
    Article Snippet: .. For the SBT3.3m-GFP construct, pDONR207+SBT3.3 vector was amplified using Phusion Hot Start II polymerase (Thermo Scientific) with SBT3.3m FW and SBT3.3m RV phosphorylated primers including a T663 to G663 mutation. .. The PCR product was then digested with DpnI restriction enzyme (Fermentas), purified by Zymoclean DNA Recovery Kit (Zymo Research) and religated using T4 Ligase (Fermentas).

    Article Title: MAS promoter regulation: A role of Sry and tyrosine nitration of the KRAB domain of ZNF274 as a feedback mechanism
    Article Snippet: .. Mutations were incorporated into primers, vector amplified with those primers using Phusion Hot Start II (Thermo) and vectors ligated together using T4 ligase. .. GST pull down assays for Sry by the GST-KRAB-O construct were performed similarly to KRAB-KAP pull down experiments.

    Article Title: Highly Expandable Human iPS Cell-Derived Neural Progenitor Cells (NPC) and Neurons for Central Nervous System Disease Modeling and High-Throughput Screening
    Article Snippet: .. pTetO-mNgn2-TA-puro ( ; available at Addgene plasmid ID# 52047) pLX-304 (Addgene plasmid ID# 25890) Phusion Hot Start II DNA polymerase (ThermoFisher Scientific cat# F549L) PacI restriction endonuclease (NEB cat# R0547S) Calf intestinal alkaline phosphatase (NEB cat# M0290S) Quick Ligation kit (NEB cat# M2200S) Stbl3 E. coli strain (ThermoFisher Scientific cat# C737303) Qiagen Plasmid Maxi Kit (cat# 12162) Transactivator plasmid pFUW-M2rtTA ( , Addgene plasmid ID# 20342 Lentiviral packaging plasmid pCMV-dR8.2 dvpr ( , Addgene plasmid ID# 8455 VSV-G envelope expressing plasmid pMD2.G (Addgene plasmid ID# 12259) Humidified incubator at 37°C with 5% CO2 HEK293T cells (ATCC 293T/17) Poly-L-Lysine (Sigma cat# P5899) D10 medium (see recipe) Lipofectamine 2000 (Thermo Fisher Scientific cat# 11668-019) Opti-MEM (Gibco cat# 31985) Neural proliferation media (NPM, see recipe) POL-coated plates (see recipe) Blasticidin (Gibco cat# A11139-03) POLS-coated plates iNgn2 neural media (N3aM, see recipe) Puromycin (Sigma cat# P8833-25MG) Cytosine arabinoside (AraC; Sigma cat# C6645) .. There are numerous selection markers and many methods to insert the selection marker of choice into the inducible Ngn2 construct (pTetO-mNgn2-TA-puro).

    Software:

    Article Title: Coregulated Genes Link Sulfide:Quinone Oxidoreductase and Arsenic Metabolism in Synechocystis sp. Strain PCC6803
    Article Snippet: Vector NTI Advance 10 software (Life Technologies) was used to design cloning steps and PCR primers. .. PCR amplification of the 3,832-bp DNA fragment containing sll5035, sll5036, and slr5037 coding sequences and slr5038 was performed using the 5038Reg2SalIFw and 5038Reg2NotRev primer pair ( ) using Synechocystis genomic DNA as the template and Phusion Hot Start polymerase (Finnzymes, Sweden) according to the manufacturer's recommendations.

    Agarose Gel Electrophoresis:

    Article Title: Engineered baker’s yeast as whole-cell biocatalyst for one-pot stereo-selective conversion of amines to alcohols
    Article Snippet: Nucleic acid manipulation Plasmid DNA was prepared with the GeneJET Plasmid Miniprep Kit (Thermo Scientific, Rockford, IL, USA) and agarose gel DNA extraction was performed using QIAquick® Gel Extraction Kit (Qiagen GmbH, Hilden, Germany). .. Primers from MWG-Biotech AG (Ebersberg, Germany) and Phusion Hot Start II DNA Polymerase and dNTPs from Thermo Scientific (Rockford, IL, USA) were used for polymerase chain reactions (PCR) and performed in a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA, USA).

    Transgenic Assay:

    Article Title: An Extracellular Subtilase Switch for Immune Priming in Arabidopsis
    Article Snippet: Paragraph title: Gene constructs and transgenic lines ... For the SBT3.3m-GFP construct, pDONR207+SBT3.3 vector was amplified using Phusion Hot Start II polymerase (Thermo Scientific) with SBT3.3m FW and SBT3.3m RV phosphorylated primers including a T663 to G663 mutation.

    High Throughput Screening Assay:

    Article Title: Transcriptional networks specifying homeostatic and inflammatory programs of gene expression in human aortic endothelial cells
    Article Snippet: .. Libraries were then amplified by PCR (Phusion Hot Start II, ThermoFisher Scientific, #F549L) and purified (Zymo #D5205) for high-throughput sequencing. .. Chromatin immunoprecipitation sequencing (ChIP-seq) ChIP-seq was performed as previously described ( ).

    Lysis:

    Article Title: Transcriptional networks specifying homeostatic and inflammatory programs of gene expression in human aortic endothelial cells
    Article Snippet: RNA-seq HAECs were resuspended in RNA Lysis Buffer and RNA was extracted from cells using the Quick-RNA Micro Prep kit from ZymoResearch (Irvine, CA, #R1051), including optional DNase I treatment. mRNA was selected through poly-A isolation using Oligo d(T)25 beads (New England BioLabs, Ipswich, MA, #S1419S). .. Libraries were then amplified by PCR (Phusion Hot Start II, ThermoFisher Scientific, #F549L) and purified (Zymo #D5205) for high-throughput sequencing.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Thermo Fisher phusion hot start ii high fidelity dna polymerase
    Clustering of samples amplified with two different polymerases on a non-metric multidimensional scaling (NMDS) plot of the Bray-Curtis dissimilarities. Points are colored by applied extraction kit. The encircled pairs correspond to a single sample where each data point represents one 16 S rRNA amplification with <t>Phusion</t> Hot Start II High-Fidelity <t>DNA</t> Polymerase and the another with Platinum SuperFi DNA Polymerase. Sample pairs labeled with * were stored in PBS.
    Phusion Hot Start Ii High Fidelity Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 82 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phusion hot start ii high fidelity dna polymerase/product/Thermo Fisher
    Average 99 stars, based on 82 article reviews
    Price from $9.99 to $1999.99
    phusion hot start ii high fidelity dna polymerase - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher phusion hot start dna polymerases
    A diagram of the Hot Fusion process for single fragment cloning. Red and blue boxes on the vector and PCR product indicate the overlapping sequences (17–30 bp). T5 exonuclease removes nucleotides from the 5′ to 3′ end of double strand <t>DNA</t> molecules. <t>Phusion</t> DNA polymerase fills in gaps that are over-generated by T5 exonuclease. Annealed fragments are transformed into E. coli and the nick sites in the nucleotide chain are repaired by E. coli.
    Phusion Hot Start Dna Polymerases, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/phusion hot start dna polymerases/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    phusion hot start dna polymerases - by Bioz Stars, 2020-04
    99/100 stars
      Buy from Supplier

    Image Search Results


    Clustering of samples amplified with two different polymerases on a non-metric multidimensional scaling (NMDS) plot of the Bray-Curtis dissimilarities. Points are colored by applied extraction kit. The encircled pairs correspond to a single sample where each data point represents one 16 S rRNA amplification with Phusion Hot Start II High-Fidelity DNA Polymerase and the another with Platinum SuperFi DNA Polymerase. Sample pairs labeled with * were stored in PBS.

    Journal: Scientific Reports

    Article Title: The impact of storage buffer, DNA extraction method, and polymerase on microbial analysis

    doi: 10.1038/s41598-018-24573-y

    Figure Lengend Snippet: Clustering of samples amplified with two different polymerases on a non-metric multidimensional scaling (NMDS) plot of the Bray-Curtis dissimilarities. Points are colored by applied extraction kit. The encircled pairs correspond to a single sample where each data point represents one 16 S rRNA amplification with Phusion Hot Start II High-Fidelity DNA Polymerase and the another with Platinum SuperFi DNA Polymerase. Sample pairs labeled with * were stored in PBS.

    Article Snippet: Platinum SuperFi DNA Polymerase (Thermo Fisher Scientific) and the Phusion Hot Start II High-Fidelity DNA Polymerase (Thermo Fisher Scientific) were both tested for amplification.

    Techniques: Amplification, Labeling

    Identification of the exponential phase during PCR amplification of barcode sequences. A. Schematic of the strategy used to identify the transition point from exponential to linear PCR amplification. gDNA isolated from HEK293T cells transduced with the pooled shRNA library were amplified in replicate PCR reactions. A replicate reaction was stopped at each cycle from 15 to 27 cycles. Subsequently, PCR products were used as templates for SYBR qPCR reactions using nested primers targeting a common sequence (outside of the barcode region) to examine the ΔC q between cycles. B. Difference of C q obtained in the qPCR on diluted amplicons from every cycle of the Phusion HS II polymerase PCR reaction (C qN+1 −C qN ) as a function of the Phusion PCR cycle number (N). C. Gel analysis of the PCR product generated from amplification cycles 22 to 25. Sizes of DNA bands in DNA marker (lane M) are indicated on the left.

    Journal: PLoS ONE

    Article Title: Optimized PCR Conditions and Increased shRNA Fold Representation Improve Reproducibility of Pooled shRNA Screens

    doi: 10.1371/journal.pone.0042341

    Figure Lengend Snippet: Identification of the exponential phase during PCR amplification of barcode sequences. A. Schematic of the strategy used to identify the transition point from exponential to linear PCR amplification. gDNA isolated from HEK293T cells transduced with the pooled shRNA library were amplified in replicate PCR reactions. A replicate reaction was stopped at each cycle from 15 to 27 cycles. Subsequently, PCR products were used as templates for SYBR qPCR reactions using nested primers targeting a common sequence (outside of the barcode region) to examine the ΔC q between cycles. B. Difference of C q obtained in the qPCR on diluted amplicons from every cycle of the Phusion HS II polymerase PCR reaction (C qN+1 −C qN ) as a function of the Phusion PCR cycle number (N). C. Gel analysis of the PCR product generated from amplification cycles 22 to 25. Sizes of DNA bands in DNA marker (lane M) are indicated on the left.

    Article Snippet: For the cell viability screen in HEK293T cells, amplification of the barcode region for microarray analysis was performed using Phusion® Hot Start II High-Fidelity DNA Polymerase (Thermo Scientific, Vantaa, Finland) and Decode negative selection primers.

    Techniques: Polymerase Chain Reaction, Amplification, Isolation, Transduction, shRNA, Real-time Polymerase Chain Reaction, Sequencing, Generated, Marker

    Clustering of samples amplified with two different polymerases on a non-metric multidimensional scaling (NMDS) plot of the Bray-Curtis dissimilarities. Points are colored by applied extraction kit. The encircled pairs correspond to a single sample where each data point represents one 16 S rRNA amplification with Phusion Hot Start II High-Fidelity DNA Polymerase and the another with Platinum SuperFi DNA Polymerase. Sample pairs labeled with * were stored in PBS.

    Journal: Scientific Reports

    Article Title: The impact of storage buffer, DNA extraction method, and polymerase on microbial analysis

    doi: 10.1038/s41598-018-24573-y

    Figure Lengend Snippet: Clustering of samples amplified with two different polymerases on a non-metric multidimensional scaling (NMDS) plot of the Bray-Curtis dissimilarities. Points are colored by applied extraction kit. The encircled pairs correspond to a single sample where each data point represents one 16 S rRNA amplification with Phusion Hot Start II High-Fidelity DNA Polymerase and the another with Platinum SuperFi DNA Polymerase. Sample pairs labeled with * were stored in PBS.

    Article Snippet: Platinum SuperFi DNA Polymerase (Thermo Fisher Scientific) and the Phusion Hot Start II High-Fidelity DNA Polymerase (Thermo Fisher Scientific) were both tested for amplification.

    Techniques: Amplification, Labeling

    A diagram of the Hot Fusion process for single fragment cloning. Red and blue boxes on the vector and PCR product indicate the overlapping sequences (17–30 bp). T5 exonuclease removes nucleotides from the 5′ to 3′ end of double strand DNA molecules. Phusion DNA polymerase fills in gaps that are over-generated by T5 exonuclease. Annealed fragments are transformed into E. coli and the nick sites in the nucleotide chain are repaired by E. coli.

    Journal: PLoS ONE

    Article Title: Hot Fusion: An Efficient Method to Clone Multiple DNA Fragments as Well as Inverted Repeats without Ligase

    doi: 10.1371/journal.pone.0115318

    Figure Lengend Snippet: A diagram of the Hot Fusion process for single fragment cloning. Red and blue boxes on the vector and PCR product indicate the overlapping sequences (17–30 bp). T5 exonuclease removes nucleotides from the 5′ to 3′ end of double strand DNA molecules. Phusion DNA polymerase fills in gaps that are over-generated by T5 exonuclease. Annealed fragments are transformed into E. coli and the nick sites in the nucleotide chain are repaired by E. coli.

    Article Snippet: Phusion Hot Start DNA polymerases and GeneRuler 1 kb Plus DNA Ladder were purchased from Thermo Scientific (Waltham, MA).

    Techniques: Clone Assay, Plasmid Preparation, Polymerase Chain Reaction, Generated, Transformation Assay