Materials Required:

  1. Bacmid DNA
  2. Abx-free ESF921 Media
  3. Sf9 cells
  4. Sterilized microcentrifuge and 50mL conical tubes

General Notes:

Be certain to count cells properly and thoroughly. Be certain to mix all reagents well. Start ASAP in morning; this is a long procedure. DO NOT interact with bacteria before going into the hood. DO maintain sterile protocol while in the hood. Bacmid DNA should be as fresh as possible. Wednesdays are ideal days for this protocol. Make CERTAIN CellfectinII is well resuspended.

Protocol Outline:

Bacmid transfection protocol is composed from my own notes (SJK) and page 14 of Preparation of the Transient Receptor Potential Vanilloid 2 (TRPV2) channel for structural studies (LZ, 2021).

  1. After PCR validation of constructs, measure nucleic acid concentration using nanodrop. Record values. Calculate 40ug of DNA. Example:

| Ortholog | Measured Conc (ug/mL) | [a] | 40 ug/uL (40/a) | [b] | Abx-Free [75-b] | | --- | --- | --- | --- | | Protein 1 | 8.1 | 4.9 uL | 70.1 uL | | Protein 2 | 7.4 | 5.4 uL | 69.6 uL | | Protein 3 | 10.0 | 4 uL | 71 uL |

  1. Calculate amount of Cellfectin transfection reagent required. Each reaction requires 6uL of Cellfectin II and 69uL of Abx-free media. Be certain to use Abx-free media as abx-media will interfere with transfections. This example has three reactions; however I make an extra reaction just in case.

6uL Cellfectin x 4 = 24uL Cellfectin reagent

69uL Abx-free media x 4 = 276uL Abx free media

  1. Each transfection requires 2mL of sf9 cells at 0.8M/mL density (1.6M cells per transfection). Calculate number of cells required from your stock, with an extra reaction just for good measure. Example:

    Passage Stock #52 has a cell density of 10M/mL

    4 transfections *2mL = 8mL final volume, 8mL * 0.8M cells = 6.4M cells required.

    6.4M required / 10M stock density = 640uL of cell stock + 7.36mL abx-free media.

  2. Record values from 1a, 2a, and 3a. Do not bring in notepads or writing utensils into tissue culture room. Rather, write the values on a paper towel and tape to the side of the hood for reference, if needed.

  3. First, prepare cells for transfection. This requires making a stock of cells in abx-free media in a 50mL conical as described in step 3.

  4. Obtain a 6-well plate. Spray thoroughly. Do not allow plate to touch surface of hood, rather remove 6-well plate from wrapper as you would a serological pipette. Label surfaces. I like to make two extra