Stably Transfected HeLa cell line construct

Seed cells

[2014 Aug 14th 15:00pm]

Method

Seed HeLa cells in 6-well-plate (15wells at total), 200,000 cells per well. For tansfection

Procedure

1. Wash cells with PBS, trypsin digestion, and medium to stop digestion.
2. Transfer the sell suspension in 15ml centrifuge tube and centrifuge at 800rpm, fro 5 min.
3. Discard the supernatant and add 2ml complete medium to re suspend the cells.
4. Add 1ml PBS to every gap between well
5. 1:10 dilution count the cell concentration with blood counting slide. Get the concentration: 2,800,000 cells/ml. 1.15ml cell suspendion + about 31ml DMEM with 5% FBS. Mix well and add 2ml per well, shake the plate gently.

Result

The cells plate unevenly concentrated at the center of each well.

Cell transfection

[2014 Aug 15th 19:00pm]

Materials

• plasmid pBx-083 (EGFP)
• plasmid Piggybac
• plasmid Cas9+TetOn
• Lipofectamine 3000(Invitrogen)
• Opti-MEM
• HeLa cells in 6-well-plate

Method

a. 3 combinations

1. EGFP(pBx-083) & Piggybac(transposase)
2. EGFP & Cas9 & Piggybac
3. Cas9(Cas9+TetOn) & Piggybac

For the first two combination we have different ratio of EGFP to get mono-copy of EGFP in cell line.

Procedure

The official protocol of Lipofectamine 3000 (Invitrogen)

We use the reduced amout of DNA.