Day 1- 2018.07.17:
- Prepare 4 plates of 60mm of diameter with 14 coverslips of 12mm.
- Treatment with Poly-D-Lysina (diluted 1/40 with boratte buffer)
- Incubate for 1h (37ºC, 5% CO2).
- Wash with PBS 4x.
- Trypsin-EDTA a plate of HEK293.
- For every plate of 60mm: 5ml of HEK medium + the volume that correspond to 1.000.000 of cells.
- Incubate at 37ºC for 24h.
Day 2- 2018.07.18:
[NF140] =1.084microg/microL.
[NF186]=0.953microg/microL.
[MBP]= 0.435microg/microL (maxiprep from glicerolate).
[LRP1]=0.353microg/microL (directly from the DNA of Genecopeia).
*For every plate of 60mm of diameter –> 8 microg of DNA + 12 microL of Lipofectamine.
- NF140 : 1 plate
- 7.4microL of DNA in 250microL of Optimem.
- 12 microL of Lipo in 250microL of Optimem.
- NF186: 1 plate
- 8.4microL of DNA in 500microL of Optimem.
- 12 microL of Lipo in 250microL of Optimem.
- MBP: 1 plate
- 18.4microL of DNA in 250microL of Optimem.
- 12 microL of Lipo in 250microL of Optimem.
- LRP1: 1 plate
- 22.6microL of DNA in 250microL of Optimem.
- 12 microL of Lipo in 250microL of Optimem.
– Incubate at RT for 5 min.
– Unify every DNA with the appropriate amount of Lipo and incubate at RT for 30 min.
– For every plate: 5ml of HEK medium and 500microL of every mix of DNA and Lipo.
– Incubate O/N at 37ºC.
Day 3- 2018.07.19:
- Fix with PFA 4%. Incubation at RT during 10min.
- Wash with PBS 1x.
- Blocking solution: 5% goat serum in PBS for 1h at RT.
- Remove the blocking solution and freeze at -80ºC.
Check the transfection – ICC:
For MBP and LRP1:
- Blocking solution: 5% goat serum in PBS
- Primary antibody: c-myc diluted 1/100
- Secondary antibody: GAM488 diluted 1/1000
- Vectashield mounting medium
For NF186 and NF140:
- Blocking solution: 5% Goat serum in PBS.
- Primary antibody: AF3235 Diluted 1/1000
- Secondary antibody: GAC488 diluted 1/1000
- Vectashield mounting medium
Results: The samples for MBP, NF186 and NF140 were well transfected. The samples transfected with LRP1 were not well transfected. These samples (LPR1) were transfected directly with DNA from the sample of Genecopoeia without doing the transformation in E. Coli and the maxipreps. Maybe we will have to repeat the process doing the transformation with E. Coli.
