20150513_bis Set up HEK co-transfection CNTN+CASPR

Objectives

We’ll transfect HEK cells with different concentration of each plasmid in order to set up the co-transfectation of CNTN1-Human and CASPR1-Human, both vectors from Genecopoeia.

Materials

see last transfection

• CASPR1-Human (Genecopoeia): EX-M0417-M02
• CNTN1-Human (Genecopoeia): EX-A1153-M02

Methods

13.05.2015

1. Put one coverslip in each well of a 24well plate
2. Incubated 1h at 37ºC with polyDlysine 1/500
3. Wash with PBS
4. Put 400ul of HEK medium and add 50000 cells per well

14.05.2015

1. Prepare DNA for transfection and lipofectamine(1ul with 15ul of OptiMEM per well) and wait for 5min
2. Mix it and incubate it 30min
3. Change the cells medium and add 30ul of each dilution in the correct well.

Experimental design: (CNTN:CASPR)

• CNTN: 1.8ug/ul
• CASPR: 1.4ug/ul

1. 1:1 – 0.3ulCNTN + 0.4ulCASPR
2. 1:2 – 0.3ulCNTN + 0.8ulCASPR
3. 1:3 – 0.3ulCNTN + 1.2ulCASPR
4. 1:4 – 0.3ulCNTN + 1.6ulCASPR
5. 1:5 – 0.3ulCNTN + 2ulCASPR
6. CNTN 0.4ul
7. 1:1 (x2) – 0.6ulCNTN + 0.8ulCASPR
8. 2:1 – 0.6ulCNTN + 0.4ulCASPR
9. 3:1 – 0.9ulCNTN + 0.4ulCASPR
10. 4:1 – 1.2ulCNTN + 0.4ulCASPR
11. 5:1 – 1.5ulCNTN + 0.4ulCASPR
12. CASPR 0.4ul

Results & Conclusions

There are cells transfected with CNTN vector and also with CASPR one.

The transfection with more cells CO-transfected is coverslip num 8 (2:1 –> 0.6ulCNTN + 0.4ulCASPR)