phusion (New England Biolabs)

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Name:

Phusion GC Buffer Pack

Description:

Phusion GC Buffer Pack 6 0 ml

Catalog Number:

B0519S

Price:

Category:

Buffers

Size:

6 0 ml

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New England Biolabs phusion

Phusion GC Buffer Pack 6 0 ml
https://www.bioz.com/result/phusion/product/New England Biolabs
Average 99 stars, based on 1 article reviews
Price from $9.99 to $1999.99

phusion - by Bioz Stars, 2021-05

99/100 stars

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1) Product Images from "RF-Cloning.org: an online tool for the design of restriction-free cloning projects"

Article Title: RF-Cloning.org: an online tool for the design of restriction-free cloning projects

Journal: Nucleic Acids Research

doi: 10.1093/nar/gks396

Figure Legend Snippet: RF-Cloning.org output page. (1) A unique 32 byte hash code is generated for all new projects, and is present in the URL for bookmarking purposes. (2) The hybrid primers are color coded, blue for sequence complementary to the plasmid, and green for the insert. The length of the primers can be adjusted by clicking on the arrow buttons if the user wishes to alter the annealing temperature. (3) If the insert site needs to be adjusted, the user can use the provided arrow buttons. (4) The secondary PCR conditions are optimized for iProof or Phusion as the polymerase, so the user should follow manufacturer’s instructions if using another high fidelity enzyme. ‘Insert’ refers to the mega-primer purified from the primary PCR reaction. (5) The entire sequence of the new plasmid is output, with insert in green and parental plasmid in blue. (6) The plasmid map can be drawn by specifying the positions of markers manually, or by auto-finding common features. Restriction enzyme cut sites can also be specified or automatically identified. If desired, the plasmid can be exported as a genbank file. (7) All projects are automatically saved, but making changes to the output page will activate the save button so those changes can be uploaded to the database. If the user has registered an account to access the plasmid management system, the save button will attach the project to their profile. (8) After the project has been completed and sent for sequencing, the sequencing results can be copied into a popup window for BLAST2 sequence alignment.

Techniques Used: Clone Assay, Polyacrylamide Gel Electrophoresis, Generated, Sequencing, Plasmid Preparation, Polymerase Chain Reaction, Purification

Polymerase Chain Reaction:

Article Title: RF-Cloning.org: an online tool for the design of restriction-free cloning projects
Article Snippet: RECOMMENDED RF-CLONING PROTOCOL By default, the starting hybrid primers will be at least 40 bp long with an annealing temperature of at least 55°C for the primary PCR (amplification of insert) and at least 60°C for the secondary PCR (extension around the plasmid). .. High-fidelity DNA polymerase should be used for all PCR reactions, and it has been our experience that iProof (BioRad, Hercules, CA, USA) and Phusion (New England Biolabs, Ipswich, MA, USA) produce consistent results. .. To generate the mega-primer, use a standard 50 μl PCR reaction (1× PCR buffer, 200 μM dNTP, 500 nM of each primer, 1 U polymerase, user defined amount of starting template) and cycle 30–35 times with the RF-Cloning.org recommended annealing temperature, followed by product purification (e.g. by gel extraction).

Article Title: High-throughput mutagenesis using a two-fragment PCR approach
Article Snippet: When using KOD Hot Start Master Mix, the following thermocycling setup was used: Initial denaturation at 95 °C for 2 min, followed by 20 step-down thermal cycles, each comprising denaturation at 95 °C for 20 s, annealing from 65 °C down to 55.5 °C for 10 s (0.5 °C decrement per cycle), and elongation at 70 °C for at the appropriate time (up to 135 s), then 5 thermal cycles with the constant annealing temperature (denaturation at 95 °C for 20 s, annealing at 62.5 °C for 10 s, elongation at 70 °C with the same time period as used in the step-down cycles), completed with a hold at 10 °C. .. For truncations of the AtETR1 and LeETR1 plasmids by the two-fragment approach, a slightly different PCR protocol was used: 20 μL PCR mixture prepared with nuclease-free water (Cell Signaling Technology) contained 1 ng DNA template, 500 nM each primer, 200 μM each dNTP (deoxynucleoside triphosphate; NEB), 0.4 U μL−1 Phusion High-Fidelity DNA Polymerase (NEB), and either 1× Phusion buffer HF (NEB) or 3% (v/v) DMSO (NEB) with 1× Phusion buffer GC (NEB). .. Also, PCR thermocycling described above was modified in case of the ETR1 plasmids to have: (1) 11 step-down cycles with annealing starting from 65 °C down to 60 °C (i.e. decreasing by 0.5 °C in each subsequent cycle); (2) elongation time of at least 30 s per kbp of the target product (up to 200 s for the longest PCR products); and (3) final elongation time of 5 min.

Article Title: Restriction enzyme-free mutagenesis via the light regulation of DNA polymerization
Article Snippet: It is important to note that the selection of primers is key, and for some sequences, longer hybridization arms are required (see GFP deletion in ), which in turn requires additional caging groups to prevent undesirable hybridization during the PCR (see ‘Results and Discussion’ section). .. The forward and reverse primers (5 μl each of a 10 μM solution), the pGFPuv template (1 μl of a 0.1 ng/μl solution), dNTPs (2 μl each of a 1 mM solution), Phusion GC Buffer (5 μl of a 10× stock, New England Biolabs), Phusion DNA Polymerase (1 μl, two units) and dH2 O (31 μl) were mixed and subjected to the following PCR program: 95°C (2 min), followed by 40 cycles of 95°C (30 s), 40°C (60 s) and 72°C (3.3 min), with a final extension at 72°C (2 min). .. An identical PCR reaction was then repeated, using 5 μl of the previous reaction as the template, followed by purification with a PCR cleanup kit (Promega).

Article Title: Effects of wine-cap Stropharia cultivation on soil nutrients and bacterial communities in forestlands of northern China
Article Snippet: .. PCR-based amplifications were performed using Phusion® High-Fidelity PCR Master Mix with GC Buffer and high-fidelity DNA polymerase (New England Biolabs, Ipswich, MA, USA) following an amplification programme of 1 cycle at 98 °C for 1 min, 30 cycles composed of three steps for each cycle (98 °C for 10 s, 50 °C for 30 s, and 72 °C for 30 s), and a final elongation step of 72 °C for 5 min. . ..

Article Title: In situ 10-cell RNA sequencing in tissue and tumor biopsy samples
Article Snippet: Poly(A) PCR Poly(A) PCR was carried out with several modifications to the earlier procedure , . .. To each tailed sample, 90 µl of poly(A) PCR buffer was added to a final concentration of 1x ThermoPol buffer (New England Biolabs), 2.5 mM MgSO4 , 1 mM dNTPs (Roche), 100 µg/ml BSA (Roche), 3.75 U Taq polymerase (NEB) and 1.5 U Phusion (NEB) and 2.5 µg AL1 primer (ATTGGATCCAGGCCGCTCTGGACAAAATATGAATTCTTTTTTTTTTTTTTTTTTTTTTTT). .. Each reaction was split into three thin-walled 0.2 ml PCR tubes and amplified according to the following thermal cycling scheme: four cycles of 1 min at 94 °C (denaturation), 2 min at 32 °C (annealing) and 2 min plus 10 sec per cycle at 72 °C (extension); 21 cycles of 1 min at 94 °C (denaturation), 2 min at 42 °C (annealing) and 2 min 40 sec plus 10 sec per cycle at 72 °C (extension).

Hybridization:

Article Title: Solid-phase cloning for high-throughput assembly of single and multiple DNA parts
Article Snippet: .. Extension An extension mix containing 17.25 μl water, 2.5 μl Phusion buffer (10×), 2.5 μl dNTPs (2 mM) and 0.25 μl Phusion (2 U/ μl New England Biolabs, Ipswich, MA, USA) was pre-heated at 65°C before used to resuspend the beads from the hybridization. .. Phusion HF was used as preferred buffer for extension unless the insert was PCR amplified using a different buffer; in those instances the matching buffer (e.g. Phusion GC buffer) was used also during the extension.

phusion (New England Biolabs)

Structured Review

Images

1) Product Images from "RF-Cloning.org: an online tool for the design of restriction-free cloning projects"

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