IGEM:University of Debrecen:Transfection protocol

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Transfection protocol with PEI nuclear receptor construct

 

 

 

Background

The following protocol has been used to succesfully introduce foreign plasmid DNA into pluripotent cells through PEI mediated transfection.

 

Overwiew

Performing the protocol from beginning to end takes approximately two days and it is divided into two major procedures:

1.     Plating COS1, 293T etc. Cells into 48 (or other) well plates in order to get 60% confluency of adhaerent cells

2.     PEI mediated transfection

An elaboration of each part is found in the followings.

 

Plating COS1 and 293T cells

The goal is to get 60% confluency of adhaerent cells on a 48 well plate to transfect them.

Materials required:

  • 48 (or other) well plates
  • DMEM Medium containing 10% FBS
  • Trypsin-EDTA
  • 1% PBS
  • Bürker-chamber, centrifuge tubes, serological pipettes, pipettes and tips, vacuum aspirator and Pasteur pipettes etc.
  • Sterile laminar air flow box, incubators

Steps:

1.     Prepare the sterile box, and prewarm the medium, trypsin-EDTA and PBS up to 370C.

2.     Get the cells to the sterile box in a closed cell culturing flask or Petri dish. Open it only under the sterile box!

3.     Gently remove used medium from the cells using vacuum aspirator with Pasteur pipette.

4.     Wash the cells with 2-3 ml PBS, then remove it gently by vacuum aspirator with Pasteur pipette.

5.     Incubate the Cos1 cells 5 mins (the 293T cells 2 mins) with trypsine-EDTA at 370C, then check them under phase-contrast microscope whether they are detached from the surface.

6.     Add 4 ml medium to the trypsinised cells, resuspend them and check them under phase-contrast microscope if you get individual cells.

7.     Prepare the Bürker-chamber and do a cell count: Pipette 10 µl cell suspension into the chamber, then count 3 squares lined with 3 paralells and take their average. If it is X, you have 10000*X cells/ 1ml medium. We need 1,5 million cells/ whole plate (In the case of 48 well plates it is appr. 30000 cells/ well).

8.     Put the needed amount of the cell suspension to a 50 ml centrifuge tube, dilute it with 10% FBS DMEM, and resuspend them (In the case of 48 well plates you need 200 µl diluted cell suspension/ well).

9.     Pipette the distinct amount of cell suspension to each well, close the plate, then gently swirl it. Check the cells under microscope.

10.  Incubate the cells for 1 day at 370C, 5% CO2.

 

PEI mediated transfection

The goal is to introduce foreign plasmid DNA into COS1 and 293T cells

Materials required:

  • Plated cells
  • DMEM Medium containing 1% FBS
  • 100 mM PEI
  • 150 mM NaCl
  • TE –buffer (filtered)
  • Plasmids: Beta-Gal, Luciferase, VDR+, RXR-Full length, VDR-1, PPARgamma-LBD (their concentrationhas been measured previously)
  • Eppendorf tubes, centrifuge tubes, serological pipettes, pipettes and tips, vacuum aspirator and Pasteur pipettes etc.
  • Sterile laminar air flow box, incubators

Steps:

1.     Prepare the sterile box, and prewarm the medium up to 370C.

2.     Change the medium at least 1 hour before transfection to DMEM containing 1% FBS. (gently remove used medium from the cells using vacuum aspirator with Pasteur pipette, then pipette 200 µl DMEM containing 1% FBS)

3.     Mix gently plasmids untill they get homogenous (Do not vortex!)

4.     Dilute plasmids to 0,1 µg/µl cc. with TE-buffer in sterile Eppendorf tubes. (First count how many folds dilution is needed, then take 1 part plasmid and needed fold-1 part TE buffer)

5.     Prepare DNA mixes:

Vitamin D-RXR construct:

<tbody> </tbody>

µg DNA

Per 1 well

Total plate

VDR+RXR

VDR+(VDR-1)

RXR+(VDR-1)

2X(VDR-1)

well no

1

72

16

16

16

16

B-Gal

0.18

12.96

 

 

 

 

Luc

0.23

16.56

 

 

 

 

Mastermix 1

0.41

29.52

6.6

6.6

6.6

6.6

Receptor1

0.05

 

0.7

0.7

0.7

0.7

Receptor2

0.05

 

0.7

0.7

0.7

0.7

Total plasmid DNA

0.5

 

8

8

8

8

Create Mastermix 1 for the whole plate (129,6 µl B-gal+ 165,6 µl Luc), then put 66 µl Mastermix 1 into four Eppendorf tubes. The plasmids of special receptors have to be added to each Eppendorf tubes (7 µl from each)

PPAR gamma construct:

<tbody> </tbody>

B-gal

180ng/well

Luc

230ng/well

PPARg-LBD

120ng/well

 

6.     Add 25 µl 150 mM NaCl solution/well to each DNA mix, then vortex briefly.

7.     Prepare PEI mix: add 1,3 µl PEI solution to 25 µl 150 mM NaCl/well, then vortex briefly.

8.     Add PEI mix to DNA mixes in drops: 25 µl PEI mix 25 µl DNA mix/well, then vortex briefly. (Do not add DNA mixes to PEI mix!)

9.     Incubate transfection mixes for 20 mins at room temperature.

10.  Add 50 µl transfection mix to each well in drops, then swirl gently the plates.

Vitamin D-RXR construct:

<tbody> </tbody>
<img height="180" src="javascript:void(0);" width="137">

 


                                                                                     (VDR-1)X2

                                                                                     VDR+VDR-1

                                                                                     RXR+VDR-1

                                                                                     VDR+RXR

                                                                                     (VDR-1)X2

                                                                                     VDR+VDR-1

                                                                                     RXR+VDR-1

                                                                                     VDR+RXR

 

11.  Incubate the cells for 5-6 hours at 370C, 5% CO2.

 




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