Cart $0.00 (0)
Distributors
Careers
 
 

Applications: Co-transfection

Co-transfection of Plasmid DNA and mRNA

Co-delivery of plasmid DNA and mRNA is a co-transfection technique employed in specific applications such as creating gene insertion using Zinc Finger Nucleases (ZFNs). Following is a straight-forward protocol using TransIT®-mRNA Transfection Kit adapted from Sigma's user manual for CompoZr® Targeted Integration Kit-AAVS (Sigma Aldrich). While this co-transfection protocol has been reported to work with TransIT®-mRNA Transfection Kit, other Mirus' DNA transfection reagents deliver DNA more efficiently. For optimal co-delivery of plasmid DNA and mRNA, we recommend making separate DNA transfection complexes using one of Mirus' broad spectrum DNA transfection reagents such as TransIT-X2® Dynamic Delivery System followed by separate mRNA transfection complexes using TransIT®-mRNA Transfection Kit. This order of addition is due to the time-sensitive nature of mRNA transfection complex formation using TransIT®-mRNA Transfection Kit. Detailed protocol links for each approach are listed below.

Transient DNA & mRNA co-transfection protocol in a 6-well plate using TransIT®-mRNA Transfection Kit
Transient DNA & mRNA co-transfection protocol in a 6-Well Plate using TransIT-X2® System (for DNA delivery) and
TransIT®-mRNA Transfection Kit (for mRNA delivery)

Transient DNA & mRNA Co-Transfection Protocol in a 6-Well Plate using TransIT®-mRNA Transfection Kit

A. Plate cells

  1. Approximately 18-24 hours before transfection, plate cells using the following guidelines. For most cell types, cultures should be ≥80% confluent at the time of transfection. For adherent cells, plate cells at a density of 2-6 × 105 cells/well. For suspension cells, plate cells at a density of 8-10 × 105 cells/well.
  2. Incubate the cells overnight.

B. Prepare co-transfection complexes using TransIT®-mRNA Kit

  1. Warm TransIT®-mRNA and mRNA Boost reagents to room temperature and vortex gently before using.
  2. Place 250 μl of Opti-MEM® I Reduced Serum Medium in a sterile tube.
  3. Add 2.5 μg (2.5 μl of a 1 μg/μl stock) RNA. Pipet gently to mix completely.
  4. Add 2.5-10 µg DNA; this might be application specific. For gene integration using CompoZr® Targeted Integration Kit -AAVS1, 8.75 µg (5 μl of a 1.75 μg/μl stock) is recommmended as per manufacturer's protocol.
  5. Add 2.5-7.5 μl mRNA Boost Reagent to the diluted RNA and DNA mixture. Pipet gently to mix completely.
  6. Add 2.5-7.5 μl TransIT®-mRNA Reagent to the diluted RNA and DNA mixture. Pipet gently to mix completely.
  7. Incubate at room temperature for 2-5 minutes to allow sufficient time for complexes to form. Do not incubate the complexes for more than 5 minutes.

NOTE: Depending on the application, the optimal reagent levels to use for RNA and DNA co-transfection should be tested by including a range of TransIT®-mRNA Reagent levels (2.5,5, and 7.5 μl) with a range of mRNA Boost Reagent amounts (2.5, 5, and 7.5 μl). The amounts of RNA and DNA themselves might also need to be optimized to ensure successful co-delivery.

C. Distribute the complex mixtures to cells in complete growth medium

  1. Add the co-transfection complexes (prepared in Step B) drop-wise to different areas of the wells.
  2. Gently rock the culture vessel back-and-forth and from side-to-side to evenly distribute the co-transfection complexes.
  3. Incubate for 24-72 hours or as required. It is not necessary to replace the complete growth medium with fresh medium.
  4. Harvest cells and assay.

NOTE: While the above protocol has been reported to work with TransIT®-mRNA Transfection Kit, we recommend making separate DNA transfection complexes using one of Mirus' broad spectrum DNA transfection reagents such as TransIT-X2® Dynamic Delivery System followed by separate mRNA transfection complexes using TransIT®-mRNA Transfection Kit. This allows optimal co-delivery of plasmid DNA and mRNA; a detailed protocol is listed below.

Transient DNA & mRNA Co-Transfection Protocol in a 6-Well Plate using
TransIT-X2® Dynamic Delivery System and TransIT®-mRNA Transfection Kit

A. Plate cells

  1. Approximately 18-24 hours before transfection, plate cells using the following guidelines. For most cell types, cultures should be ≥80% confluent at the time of transfection. For adherent cells, plate cells at a density of 2-6 × 105 cells/well. For suspension cells, plate cells at a density of 8-10 × 105 cells/well.
  2. Incubate the cells overnight.

B. Prepare DNA transfection complexes using TransIT-X2® Dynamic Delivery System
(Immediately before transfection)

  1. Warm TransIT-X2® to room temperature and vortex gently before using.
  2. Place 250 µl of Opti-MEM® I Reduced-Serum Medium in a sterile tube.
  3. Add 2.5 µg (2.5 µl of 1 µg/µl stock) plasmid DNA. Pipet gently to mix completely.
  4. Add 7.5 µl of TransIT-X2®. Pipet gently to mix completely. For further optimization of your cell type, test additional amounts of TransIT-X2®.
  5. Incubate at room temperature for 15-30 minutes to allow sufficient time for complexes to form.

C. Prepare mRNA transfection complexes using TransIT®-mRNA Kit
(while the DNA transfection complexes are incubating)

  1. Warm TransIT®-mRNA and mRNA Boost reagents to room temperature and vortex gently before using.
  2. Place 250 μl of Opti-MEM® I Reduced Serum Medium in a sterile tube.
  3. Add 2.5 μg (2.5 μl of a 1 μg/μl stock) RNA. Pipet gently to mix completely.
  4. Add 5 μl mRNA Boost Reagent to the diluted RNA mixture. Pipet gently to mix completely.
  5. Add 5 μl TransIT®-mRNA Reagent to the diluted RNA mixture. Pipet gently to mix completely.
  6. Incubate at room temperature for 2-5 minutes to allow sufficient time for complexes to form. Do not incubate the complexes for more than 5 minutes.

D. Distribute the complex mixtures to cells in complete growth medium

  1. Add the co-transfection complexes (prepared in Step B and C) drop-wise to different areas of the wells.
  2. Gently rock the culture vessel back-and-forth and from side-to-side to evenly distribute the co-transfection complexes.
  3. Incubate for 24-72 hours or as required. It is not necessary to replace the complete growth medium with fresh medium.
  4. Harvest cells and assay.

Technical Resources

Reagent Agent Don't See Your Cell Type? Consult Reagent Agent® Transfection Database
Citation Database: Check if our reagents have been used by other researchers to transfect your cell type
Technical Support: Speak directly with a transfection expert