We made a ligation reaction.
prepare two tubes, one with H2O, Buffer, vector, insert and ligase, and the other the same but without the insert, this will serve to verify that the two ends of the vector are not back together. We leave in a water bath at 26 ° C for one hour to correctly insert is linked to the vector. We then proceeded with the following protocol: Transformation of E.
coli
1. Add 100 ml of competent cells and 20 ml of ligation rx a sterile Eppendorf. Make control with 100 ml of competent cells and 0.5 ml of control plasmid (Qiagen kit purified x)
2. Incubate 30 minutes on ice
3. Incubate 2 minutes to 42 ˚ C
4. Incubate 5 min on ice
5. Add 1 ml (or 450 ml) of LB medium without ampicillin
6. Incubate 1 hour at 37 ˚ C to 225 rpm
7. Centrifuge cells and resuspend the pellet in remaining supernatant
8. Sowing the entire contents of each tube on plates with LB medium + AmpicilinaColocar in oven at 37 ˚ C
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