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While designing the mutagenic primers for substitution, my final primer sequences have two mismatches with the template for the necessary mutation to be present in the final sequence. For e.g.,...
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Incubation after inoculation: 1 hr at 37 degree celsius in a non vented t25 flask The volume of inoculum: 500 μl After incubation, I add 5 ml 2% MEM and incubate it for 4-5 days at 35 degree celsius.
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I have done 3 blind passages to concentrate the viral stock in the supernatant without adding TPCK Trypsin. After passage 1 my Ct value for INF A was 36.96. After P2 the value decreased to 33.36....
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