IGEM:University of Debrecen:Transfection protocol

 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. </li>  Sterile laminar air flow box, incubators </li> </ul>

Steps:

<p style="margin-left:45.0pt"> 1.<span style="font:7.0pt &quot;Times New Roman&quot;">     Prepare the sterile box, and prewarm the medium, trypsin-EDTA and PBS up to 370C.

<p style="margin-left:45.0pt"> 2.<span style="font:7.0pt &quot;Times New Roman&quot;">     Get the cells to the sterile box in a closed cell culturing flask or Petri dish. Open it only under the sterile box!

<p style="margin-left:45.0pt"> 3.<span style="font:7.0pt &quot;Times New Roman&quot;">     Gently remove used medium from the cells using vacuum aspirator with Pasteur pipette.

<p style="margin-left:45.0pt"> 4.<span style="font:7.0pt &quot;Times New Roman&quot;">     Wash the cells with 2-3 ml PBS, then remove it gently by vacuum aspirator with Pasteur pipette.

<p style="margin-left:45.0pt"> 5.<span style="font:7.0pt &quot;Times New Roman&quot;">     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.

<p style="margin-left:45.0pt"> 6.<span style="font:7.0pt &quot;Times New Roman&quot;">     Add 4 ml medium to the trypsinised cells, resuspend them and check them under phase-contrast microscope if you get individual cells.

<p style="margin-left:45.0pt"> 7.<span style="font:7.0pt &quot;Times New Roman&quot;">     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).

<p style="margin-left:45.0pt"> 8.<span style="font:7.0pt &quot;Times New Roman&quot;">     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).

<p style="margin-left:45.0pt"> 9.<span style="font:7.0pt &quot;Times New Roman&quot;">     Pipette the distinct amount of cell suspension to each well, close the plate, then gently swirl it. Check the cells under microscope.

<p style="margin-left:45.0pt"> 10.<span style="font:7.0pt &quot;Times New Roman&quot;">  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:

<ul style="margin-top:0in">  Plated cells </li>  DMEM Medium containing 1% FBS </li>  100 mM PEI </li>  150 mM NaCl </li>  TE –buffer (filtered) </li>  Plasmids: Beta-Gal, Luciferase, VDR+, RXR-Full length, VDR-1, PPARgamma-LBD (their concentrationhas been measured previously) </li>  Eppendorf tubes, centrifuge tubes, serological pipettes, pipettes and tips, vacuum aspirator and Pasteur pipettes etc. </li>  Sterile laminar air flow box, incubators </li> </ul>

Steps:

<p style="margin-left:45.0pt"> 1.<span style="font:7.0pt &quot;Times New Roman&quot;">     Prepare the sterile box, and prewarm the medium up to 370C.

<p style="margin-left:45.0pt"> 2.<span style="font:7.0pt &quot;Times New Roman&quot;">     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)

<p style="margin-left:45.0pt"> 3.<span style="font:7.0pt &quot;Times New Roman&quot;">     Mix gently plasmids untill they get homogenous (Do not vortex!)

<p style="margin-left:45.0pt"> 4.<span style="font:7.0pt &quot;Times New Roman&quot;">     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)

<p style="margin-left:45.0pt"> 5.<span style="font:7.0pt &quot;Times New Roman&quot;">     Prepare DNA mixes:

<p style="margin-left:27.0pt"> Vitamin D-RXR construct:

<p style="margin-left:27.0pt"> 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)

<p style="margin-left:27.0pt"> PPAR gamma construct:

<p style="margin-left:45.0pt"> 6.<span style="font:7.0pt &quot;Times New Roman&quot;">     Add 25 µl 150 mM NaCl solution/well to each DNA mix, then vortex briefly.

<p style="margin-left:45.0pt"> 7.<span style="font:7.0pt &quot;Times New Roman&quot;">     Prepare PEI mix: add 1,3 µl PEI solution to 25 µl 150 mM NaCl/well, then vortex briefly.

<p style="margin-left:45.0pt"> 8.<span style="font:7.0pt &quot;Times New Roman&quot;">     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!)

<p style="margin-left:45.0pt"> 9.<span style="font:7.0pt &quot;Times New Roman&quot;">     Incubate transfection mixes for 20 mins at room temperature.

<p style="margin-left:45.0pt"> 10.<span style="font:7.0pt &quot;Times New Roman&quot;">  Add 50 µl transfection mix to each well in drops, then swirl gently the plates.

<p style="margin-left:27.0pt"> Vitamin D-RXR construct:

<p style="line-height:150%">                                                                                    (VDR-1)X2

<p style="line-height:150%">                                                                                    VDR+VDR-1

<p style="line-height:150%">                                                                                    RXR+VDR-1

<p style="line-height:150%">                                                                                    VDR+RXR

<p style="line-height:150%">                                                                                    (VDR-1)X2

<p style="line-height:150%">                                                                                    VDR+VDR-1

<p style="line-height:150%">                                                                                    RXR+VDR-1

<p style="line-height:150%">                                                                                    VDR+RXR

<p style="margin-left:45.0pt"> 11.<span style="font:7.0pt &quot;Times New Roman&quot;"> Incubate the cells for 5-6 hours at 370C, 5% CO2.