Cloning và transformation trên vi khuẩn E.coli

CLONING AND TRANSFORMATION

1. pENTR vector and insert

- 0.5 ul pENTR/TOPO (~ 10ng)

- 0.5 ul salt solution

- 1 ul insert

 pENTR/Insert = 1:5

   2.6kb/ n         = 1:5

n ~ 29 ng of insert

- water 1 ul

V_tot = 3 ul

Mix gently and incubate at room temperature 20 min

2. Transformation

- Add 3 ul cloning reaction into vial E.coli and mix gently. DO NOT mix by pipetting up and down

- Incubate on ice 20 min

- Heat-shock 40 second at 42⁰

- Immediately transfer tube to ice

- Add 250 ul LB medium

- Cap the tube and shake horizontally (200 rpm) at 37⁰C for 2 h

- Spread transformation and incubate 37⁰C overnight

                        3. Transfering a target gene from pENTR vector to pK7FWG2

                                    - 75 ng entry vector (target gene)                   a ul

                                    - 75 ng destination vector (pK7FWG2)          b ul

                                    - LR cloning buffer                                         c ul

                                                                                                            ------

                                    V_tot                                                                  4 ul

                                    - LR clonase                                                    1 ul

                                    - Incubate 1h at 37⁰C

                                    - Add protein K (incubate 10 min)                 1 ul

                                                                                                            -----

                                    V_tot                                                                  6 ul

4. Digestion

- Buffer D                               1 ul

- Plasmid (500-1000ng)          5 ul

- H₂O                                       3 ul

- Enzyme (NotI+EcoRV)       1 ul (0.5+0.5)

                                                ---------

V_tot                                          10 ul

Incubate 37⁰C