Editing MuLV Psueduvirus (section)

=== '''Lipid-based transfection reagent mix'''&nbsp;(see&nbsp;'''Table of Materials''') ===

#Calculate the volumes for the transfection reagent mix from the quantities shown in&nbsp;'''Table 2'''&nbsp;for one well (1:3 transfection ratio, multiply quantities as needed). Include extra wells to avoid running out of transfection reagent mix.
#Mix calculated volumes of lipid-based transfection reagent (3 µL per well) and reduced serum cell culture medium (47 µL per well) in a microcentrifuge tube, making sure to add the transfection reagent into the reduced serum cell culture medium and not the other way around.
#Incubate both mixes (for one well: 50 µL of plasmids mix and 50 µL of lipid-based transfection reagent mix) separately for 5 min at room temperature.
#Add the contents of the transfection reagent mix to the plasmids mix at a 1:1 ratio (for 1 well: 50 µL of each mix)
#Perform thorough up-down pipetting of the resulting mix.
#Incubate the mix for at least 20 min at room temperature.
#Aspirate the spent medium of cells.
#Add gently 1 mL of pre-warmed (37 °C) reduced serum cell culture medium per well.
#Add dropwise 100 µL of transfection mix to each well.&nbsp;

&nbsp;'''NOTE:'''&nbsp;Exercise care when adding the transfection mix to wells of HEK-293T as they detach easily.
<ol start="10">
<li>Incubate cells in a 37 °C, 5% CO<sub>2</sub>&nbsp;cell culture incubator for 4–6 h.</li>
</ol>
<ol start="11">
<li>Add 1 mL per well of pre-warmed (37 °C) DMEM-T medium, which does not contain antibiotics</li>
</ol>

'''NOTE:'''&nbsp;This is a key step. Transfection reagents increase cell permeability and increase sensitivity to antibiotics. To ensure good transfection efficiency and pseudotyped particle production, it is important to avoid using cell culture medium containing antibiotics
<ol start="12">
<li>Incubate cells in a 37 °C, 5% CO<sub>2</sub>&nbsp;cell culture incubator for 48 h.</li>
</ol>