Phusion Site-Directed Mutagenesis Kit

Protocol tips

Upstream tips	Protocol tips	Downstream tips
Phusion Hot Start II DNA Polymerase tends to work better at elevated denaturation and annealing temperatures due to higher salt concentrations in its buffer. Hence follow manufacturer instruction strictly.

Upstream tips
Phusion Hot Start II DNA Polymerase tends to work better at elevated denaturation and annealing temperatures due to higher salt concentrations in its buffer. Hence follow manufacturer instruction strictly.

Publication protocol

Recombinant LCMV (rLCMV) was generated as previously described (21, 22). In brief, after (consensus) cDNAs of the large and small segments of LCMV (ARM strain-derived variant Cl13) were generated, both segments were separately placed under the control of a pol I expression cassette including the 5′ and 3′ untranslated regions (UTRs). The minimal viral trans-acting factors, the polymerase (L) and the nucleoprotein (NP), are expressed by a beta-actin-driven promoter. Seventy-two to 96 h after Lipofectamine plasmid transfection, infectious virus was recovered from the BHK-21 supernatant. Infecting BHK-21 cells at a multiplicity of infection (MOI) of 0.01 after 48 h yielded a working stock (rLCMV/Cl13). By PCR cloning, the GP of LCMV was exchanged to create several LCMV-Lassa virus Josiah GP-2 chimeric viruses. All Lassa virus/LCMV cytosolic domain mutants were generated by circular PCR using the high-fidelity Phusion site-directed mutagenesis kit (New England BioLabs Inc., Ipswitch, MA). Prior to transfection, the cloned plasmids were sequenced to control for desired mutations and to exclude unwanted random mutations. The rLCMV/LCMV-Lassa virus GP-2 chimeric viruses were rescued in the same way as described for rLCMV/Cl13.

The focus-forming assay used to determine viral titers has already been described elsewhere (23). We detected the rLCMV/Jos wt virus with the monoclonal antibody VL-4, which is directed against the nucleoprotein of LCMV and not the GP. LCMV strains Armstrong and variant Cl13 have already been described extensively elsewhere (3).

Full paper   Login or join for free to view the full paper.

Reviews

Discussion

Papers

Check out relevant papers found by Labettor's AI that are relevant for performing Site Directed Mutagenesis (SDM) Hamster - Point mutation BHK-21 LCMV (lymphocytic choriomeningitis virus) using Phusion Site-Directed Mutagenesis Kit from Thermo Fisher Scientific.

Manufacturer protocol

Download the product protocol from Thermo Fisher Scientific for Phusion Site-Directed Mutagenesis Kit below.

Download manufacturer protocol

Videos

Check out videos that might be relevant for performing Site Directed Mutagenesis (SDM) Hamster - Point mutation BHK-21 LCMV (lymphocytic choriomeningitis virus) using Phusion Site-Directed Mutagenesis Kit from Thermo Fisher Scientific. Please note that these videos are representative and steps or experiment specific processes must be kept in mind to expect desired results.