Dec 31, 2019

Public workspaceGeneral Transfection V.2

DOI
dx.doi.org/10.17504/protocols.io.bawuifew
  • 1Addgene
Addgene The Nonprofit Plasmid Repository
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DOI: dx.doi.org/10.17504/protocols.io.bawuifew
External link: https://www.addgene.org/protocols/transfection/
Protocol Citation: Addgene The Nonprofit Plasmid Repository 2019. General Transfection. protocols.io https://dx.doi.org/10.17504/protocols.io.bawuifew
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 31, 2019
Last Modified: December 31, 2019
Protocol Integer ID: 31412
Keywords: Cell Culture, Transfection
Abstract
This protocol is for general transfection. To see the full abstract and additional resources, visit the Addgene protocol page.



Sample Data

Legend: Lenti-X 293T cells were transfected using 1:1, 1:2, 1:3 and 1:6 ug of pRosetta:ug of PEI. The 1:2 and 1:3 ratios provided high transfection efficiencies as can be seen here by the amount of green fluorescent protein expression (green in the right panels) with a limited effect on cell growth.

Guidelines
Workflow Timeline

Day 0: Seed Lenti-X 293T cells (this cell line is optimized for production of lentiviral vectors)

Day 1 (pm): Transfect Cells

Day 2 (am): 18h post transfection - Remove media, replace with fresh media

Day 3 or more (am): Observe fluorescence, harvest cells, or perform your experiment


Materials
Reagents

  • DMEM high glucose
  • L-alanyl-L-glutamine (or alternative stable glutamine)
  • Heat-inactivated FBS
  • Low serum medium such as Opti-MEM or Opti-Pro SFM
  • Chloroquine diphosphate
  • Polyethyenimine, linear MW 25,000 Da
  • Microcentrifuge tubes
  • 10 cm dishes
  • Pipettes
  • Pipette tips
  • Hydrochloric acid
  • Sodium hydroxide
  • 0.22 um polyethersulfone filter
  • Syringes for filtering

Equipment

  • Biosafety cabinet
  • Pipetman
  • Pipettors
  • Incubator
  • Fluorescence microscope


Reagent Preparation

1. DMEM Complete: 10% v/v FBS and 4 mM L-alanyl-L-glutamine
  • To a Amount500 mL bottle of DMEM high glucose, add Amount55 mL of heat inactivated FBS and Amount11 mL of 200 mM L-alanyl-L-glutamine. Store at Temperature4 °C .

*Pro-Tip* Different brands and lots of FBS can promote or inhibit transfection. Test a variety of brands and lots of FBS to find one suitable with your protocols. FBS can be purchased already head inactivated or it can be inactivated in the lab by heating to Temperature56 °C for Duration00:30:00 .

2. 25 mM chloroquine diphosphate
  • Dissolve Amount0.129 g of chloroquine diphosphate salt into Amount10 mL of sterile water.
  • Filter sterilize through a 0.22 um filter.
  • Aliquot Amount50 µL - Amount100 µL and store at Temperature-20 °C .
  • Aliquots can be thawed and stored at Temperature4 °C prior to use. Thawed aliquots should be discarded after 1-2 months.

3. 1 mg/mL polyethylenimine, linear MW 25,000 Da (PEI)
  • Dissolve Amount100 mg of powder into Amount100 mL of deionized water.
  • While stirring, slowly add hydrochloric acid until the solution clears.
  • Check the pH of the solution
  • Use hydrochloric acid or sodium hydroxide to adjust the pH to 7.0. Typically the solution will be basic and will need adjustment with hydrochloric acid first.
Note
*Pro-Tip*
The pH of this solution will drift pretty rapidly upon addition of acid or base. Add only a few drops at a time, allow them to mix and recheck the pH to prevent over or undershooting the desired pH.

  • Allow the solution to mix for Duration00:10:00 and then recheck the pH to ensure that it has not drifted.
  • Filter the solution through a 0.22 um membrane.
  • Aliquot Amount500 µL - Amount1000 µL into sterile tubes.
  • Store the tubes at Temperature-80 °C .
  • After thawing the solution can be stored at Temperature4 °C for up to 2 months. After 2 months, discard the tube and thaw a new working stock.

Safety warnings
See SDS (Safety Data Sheet) for safety warnings and hazards.

Before start
Considerations Before You Start

  • The health of the packaging cell line is critical for obtaining high levels of virus.
  • Lenti-X 293T cells should be split 3 times a week:
- Monday: Plate 1x106 cells in a 75 cm2 flask in a volume of Amount15 mL .
- Wednesday: Plate 1x106 cells in a T75 flask in a volume of Amount15 mL .
- Friday: Plate 8x105 cells in a T75 flask in a volume of Amount15 mL .
  • Do not add antibiotics to the media.
  • The optimal mass DNA:mass PEI ratio will need to be empirically determined for each new batch of 1 mg/mL PEI prepared.
Note
There may be variation between batches of PEI depending on the user, quantities of chemical used, volumes, pH adjustment etc. Consequently, each batch needs to be validated and the best ratio of mass DNA:mass PEI determined.

Seeding cells
Seeding cells
Seed 293T packaging cells at 3.8x106 cells per plate in DMEM complete in 10 cm tissue culture plates.
Incubate the cells at Temperature37 °C , 5% CO2 for ~Duration20:00:00 .
Transfection
Transfection
Gently aspirate media, add Amount10 mL fresh DMEM complete containingConcentration25 micromolar (µM) cloroquine diphosphate and incubate ~Duration05:00:00 .
Note
For Amount10 mL of DMEM complete, add Amount10 µL of Concentration25 millimolar (mM) chloroquine diphosphate.

Dilute Amount18.9 µg of DNA into Amount500 µL of Opti-Pro SFM.
Note
*Pro-Tip*
Endotoxins can inhibit transfection, therefore, plasmid DNA purification should include an endotoxin removal step. For high quality plasmid DNA, the plasmid should also be propagated in an endonuclease negative E. coli strain such as NEB stable.

Dilute 1:3 (ug DNA:ug PEI) in Amount500 µL total of OptiPro SFM (per 10 cm plate).
Note
*Pro-Tip*
The ratio of ug DNA:ug PEI needs to be empirically determined. Once a batch of PEI is prepared, transfect cells with a fluorescent plasmid using a variety of ratios. Check the cells 1-2 days after transfection to determine what ratio gives the highest percentage of GFP positive cells.


Step 5 example:

Amount56.7 µL of 1 mg/mL PEI, MW 25,000 Da in Amount386.6 µL of OptiPro SFM per 10 cm plate.

Ratio of DNA:PEIAmount of DNA (μg)Volume of 1 mg/mL PEI (μL)
1:118.918.9
1:218.937.8
1:318.956.7
1:418.975.6
1:518.994.5
1:618.9113.4
Refer to this table for a possible range of ratios to test.

Gently add the diluted PEI to the diluted DNA. Add the diluted PEI dropwise while gently flicking the diluted DNA tube. Incubate the mixture Duration00:15:00 - Duration00:20:00 at TemperatureRoom temperature .
Gently add the diluted PEI to the diluted DNA.
Add the diluted PEI dropwise while gently flicking the diluted DNA tube.
Incubate the mixture Duration00:15:00 - Duration00:20:00 at TemperatureRoom temperature .
Carefully transfer the transfection mix to the Lenti-X 293T packaging cells. Add the transfection mix dropwise being careful not to dislodge the cells.
Incubate the cells for Duration18:00:00 , or until the following morning.
The following morning, carefully aspirate the media. Replace the media with Amount15 mL of DMEM complete.

Incubate the cells Duration24:00:00 - Duration48:00:00 before checking for protein expression.