PEI Transfection

 1. PEI Stock 만드는 법

1) 1mg/ml 되도록 PEI를 DNase, RNase Free-DW로 녹인다

2) Water bath 80도에서 2시간 가열하여 완전히 녹임

3) pH9정도 되는 것을 1M HCL로 pH6.7로 맞춤

4) 0.22microM syringe filter로 filtering

5) aliquot해서 -70도에 보관

6) 한번녹인것은재사용하지않음

2. PEI로 transfection하는 경우

만들어져 있는 PEI stock을 녹인 후

잘 흔들어 준 뒤

cell에 transfection 할 plasmid DNA 총 량 (micogram)에 곱하기 6만큼 PEI를 넣어준다

1) transfection할 plasmid DNA 튜브에 넣어준다

2) SerumFree DMEM 을 transfection할 dish에 넣어준 medium볼륨의 1/10만큼 넣어준다

3) PEI를 DNA 총량의 *6 만큼 넣는다

4) 잘 섞어 준 뒤 centrifuge로 down 시킨다

5) 10mins동안 RT에서 incubation.

6) cell에 뿌려준다.

7) 5시간 뒤 새로운 medium으로 바꿔준다. 

8) transfection한 뒤 24시간 후 cell harvest.

ex) transfection할 plasmid DNA 4micogram

     transfection할 cell dish 60mm (Medium total volume 4ml)

     Serum Free DMEM 400ul

     PEI 24ul

1. Preparation of PEI Stock Solution

1. Dissolve PEI in DNase/RNase-Free DW to a final concentration of 1 mg/mL.
2. Heat the solution in an 80°C water bath for 2 hours to ensure complete dissolution.
3. Adjust the pH to 6.7 using 1M HCl (starting from approximately pH 9).
4. Filter the solution using a 0.22 µm syringe filter.
5. Aliquot the solution and store at -70°C.
6. Do not reuse once thawed.

2. Transfection Using PEI

After thawing the prepared PEI stock, mix well before use. Add PEI at a ratio of 6 times the total plasmid DNA amount (µg) for transfection.

Protocol:

1. Add the plasmid DNA to a microcentrifuge tube.
2. Add 1/10th of the total medium volume of Serum-Free DMEM.
3. Add PEI at 6 times the total plasmid DNA amount.
4. Mix well and briefly centrifuge to collect the mixture at the bottom of the tube.
5. Incubate at room temperature for 10 minutes.
6. Add the transfection mixture to the cells.
7. After 5 hours, replace with fresh medium.
8. Harvest the cells 24 hours post-transfection.

Example Calculation:

• Plasmid DNA: 4 µg
• Cell dish: 60 mm (Total medium volume: 4 mL)
• Serum-Free DMEM: 400 µL
• PEI: 24 µL