Required ng, and the medium and reagent volumes, come from the spreadsheet for the vessel you pick. Change a stock concentration after a new miniprep and the volumes follow.
The ng column is the amount for one 10 cm dish; it is scaled for the vessel above. pmol is worked out from it and tells you whether a swapped-in plasmid still has the right molar share.
| Plasmid | bp | Stock ng/µl | ng at 10 cm | µl per reaction |
|---|
B. Prepare the TransIT-LT1:DNA complex, immediately before transfection. Warm TransIT-LT1 to room temperature and vortex gently. Put Opti-MEM in a sterile tube, add the plasmid DNA, mix gently, then add TransIT-LT1 without letting it touch the tube wall, and mix gently again. Stand 15–30 min at room temperature.
C. Distribute. Add drop-wise to different areas of the well, rock back-and-forth and side-to-side, then incubate 24–72 h. The growth medium does not need replacing.
Volume = required ng ÷ stock ng/µl. pmol = ng ÷ (bp × 0.66). Viral boost = medium ÷ 500.
Two places this deliberately departs from the spreadsheet, because the spreadsheet contradicts itself. Its packaging mastermix is a fixed 4.5 reactions' worth, and 45 on the 12-well sheet, which is the same number with the decimal point lost; this asks you how many reactions you are doing instead. And its 6-well TransIT-LT1 is 7 µl on the LV2 row but 44/6 = 7.33 µl on the other three; this uses 7.33 throughout, which keeps the same LT1-per-µg ratio as the 10 cm and 15 cm sheets.
Only the required ng scales cleanly by vessel. TransIT-LT1, Opti-MEM and the medium are the spreadsheet's own per-vessel numbers and are not exact multiples, so they are taken as given rather than scaled.
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