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Tips from the Bench: Transfection Tip

Reverse Transfection Protocol for DNA

Advances in high throughput (HT) screening as well as availability of multi-well cDNA/shRNA libraries have made reverse transfection of DNA commonplace.  All TransIT® DNA transfection reagents, including TransIT®-2020 Transfection Reagent, can be used for reverse transfection of DNA into multiple cell types. Additionally, cryopreserved cell stocks  can be utilized for immediate transfection reducing overall culture time. Experimental details can be found below.

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Reverse Transfection of Normal or Frozen Spun HEK 293 Cells Using TransIT-2020 Transfection Reagent

Reverse Transfection of Normal Trypsinized or Frozen Spun HEK 293 Cells Using TransIT®-2020 Transfection Reagent. HEK 293 cells were either trypsinized using standard cell culture techniques (normal trypsinized) or brought out from cryopreservation and centrifuged to remove the DMSO from the freezing media (frozen spun). These normal trypsinized or frozen spun HEK 293 cells were resuspended in complete media at two different cell densities (100,000 cells/well or 250,000 cells/well) and reverse transfected using TransIT®-2020 Transfection Reagent using 0.5 µg DNA per well at the indicated reagent-to-DNA ratios in a 24 well plate. Luciferase activity was measured at 24 hours post-transfection. Reverse transfection with TransIT®-2020 Transfection Reagent yields comparable luciferase expression in normal trypsinized cells as well as frozen spun cells straight out of cryopreservation. Higher protein expression is observed at higher cell densities.


Reverse Transfection Protocol for DNA in 96-well Plates Using TransIT®-2020 Transfection Reagent

Before You Start

Akin to forward transfection, optimizing transfection conditions by transfecting a reporter plasmid into the cell type of interest (such as a luciferase or GFP encoding plasmid) into cells is critical prior to high throughout screening.

  • For best results, determine the appropriate dose of DNA and transfection reagent in the multi-well format for the screen. Specific tips on optimizing plasmid DNA transfection can be found here. For example, the optimal DNA concentration generally ranges between 0.05-0.2 μg/well of a 96-well plate with an optimal TransIT®-2020 transfection reagent:DNA ratio of 1-4 μl per 1 μg DNA.
  • Observe reporter gene expression and toxicity at regular time points over at least a 24 hour period, optimally for 48 hours.

Use the optimal DNA and transfection reagent dosage for HT screening applications.

A. Cell Plating Prior to Transfection

  1. At least 24 hours prior to transfection, plate cells at an appropriate cell density in a T-75 cm2 flask or similar tissue culture dish so that the cells will be 70-80% confluent the following day. Approximately 2-6 x 106 cells will be needed per 96-well plate. Multiple flasks may need to be prepared if more than one 96-well plate is to be transfected.
  2. Incubate the cells overnight.

B. Complex Formation for 96-well Plates

  1. In each well of the 96-well plate to be used for transfection, add appropriate amount of serum-free medium (i.e. Opti-MEM® I Reduced Serum Medium) (see Table 1). Note: Alternatively, a mastermix can be prepared in a sterile tube if transfecting the same plasmid throughout the plate. Calculations are shown in Table 1 for 120 wells to account for pipetting errors. If a transfection master mix is prepared, prepare the transfection mixture as follows (Steps 1-4) and add per well to the 96-well plate using a multi-channel pipettor or liquid handler after complex formation (Step 4).
  2. Add appropriate amount of stock plasmid DNA (see Table 1) to the wells containing the each well of the 96-well plate.
  3. Add appropriate amount of TransIT®-2020 Transfection Reagent to the Opti-MEM® I plasmid DNA mixture (see Table 1).
  4. Incubate at room temperature for at least 15 minutes to allow for the transfection complexes to form. Note: If you are using preprinted cDNA/shRNA screens, allow for an additional 10 minutes for the reconstitution of the dried printed DNA. Do not allow complexes to incubate longer than 60 minutes before adding cells from step C below.

C. Cell Plating in 96-well Plates

  1. Trypsinize cells in T-75 cm2 flask (from Step A) as per standard tissue culture procedure. Note: To prevent re-adherence of the cells to the flask, perform this step no more than one hour prior to transfection. To further reduce cell culture time, cell plating can also be performed using cryopreserved cell stocks, after they are centrifuged to remove DMSO and counted using Trypan Blue.
  2. Add 5-10 ml of complete media to the cell suspension. Mix thoroughly by pipetting.
  3. Count the cells using a hemacytometer to determine the appropriate volume of cells in media to obtain 1.6-4.8 x 105 cells per ml.
  4. Add 92 µl of diluted cell mixture (1.4-4.4 x 104 cells) to each well. Gently rock the dish back and forth and from side to side to distribute the cells evenly. Do not swirl or rotate the dish, as this may result in uneven distribution.
  5. Incubate 24-48 hours.
  6. Harvest and assay for gene expression or other reporter assay.

Table 1. Recommended starting conditions for reverse DNA transfections with TransIT®-2020 Transfection Reagent

  Volume needed per well of a 96-well plate Total volume needed if preparing a mastermix for a 96-well plate*
Volume of serum free media for transfection complex formation 9 µl 9 x 120 = 1080 µl =1.08 mls
Amount of DNA needed per well ‡ 0.1 µg 0.1 x 120 = 12 µg
Volume of TransIT®-2020 Transfection Reagent per well ‡ 0.3 µl 0.3 x 120 = 36 µl
Trypsinized cells in complete growth medium 92 µl 92 x 120 = 11,040 ul = 11.04 mls

* The mastermix calculations are based on 120 wells to account for pipetting errors.

‡ If small volumes of TransIT®-2020 and plasmid DNA need to be pipetted, dilute the required volume of reagent and DNA ten-fold in Opti-MEM® I Reduced-Serum Medium before each use to avoid pipetting errors. Do not store diluted TransIT®-2020 Reagent or DNA stocks.


Citations for Reverse Transfection of DNA using TransIT® Transfection Reagents

Citation Cell Type Transfected TransIT® Reagent Used Robotic system used Multi-well format Application
Zhao et al. Molecular Neurodegeneration 2009, 4:4 HeLa TransIT®-LT1 None 384-well HT fluorescence polarization-based Aβ degradation assay
Owens et al. J Biol Chem. 2010 February 26; 285(9): 6761-6769. HeLa TransIT®-LT1 Multidrop 384 (Titan) 384-well HT cell-based screens to detect stabilized protein targets following chemical mutagenesis
Warzecha et al. Mol Cell. 2009 March 13; 33(5): 591-601. HEK 293T cell clone stably expressing the luciferase splicing reporter TransIT®-293 Wellmate Handler (Matrix) 384-well HT cell-based genome-wide cDNA expression screening

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