Brandon:Protocols/Cloning Transformation3

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cloning and A-tailing etc.

1. reaction from the manual


transfect into E. coli, use transfection protocol


1.  Thaw one vial of One Shot TOP10 chemically competent cells for each transformation
    Add 1-3 uL of PCR product to TOP10 cell vial (uL based on calculation)
    MIX GENTLY, no pipetting up and down

2.  incubate vials on ice for 30 minutes

3.  Heat-shock cells for 30 seconds at 42C, no shaking

4.  remove from 42C and place on ice for 2 minutes

5.  add 250 ul pre-warmed SOC medium to each vial

6.  cap vials tightly and shake horizontally at 37C for 1 hr at 224 rpm in shaking incubator

7.  spread 20-200 ul of transformations onto pre-warmed selective plates and incubate overnight at 37C.
    *use 2-3 different concentrations, 1:10, 50 uL, 200 uL or w/e

8.  Store reamining transforamtion mix at 4C. cells can be plated next day.

9.  Invert selective plates, incubate at 37 overnight.

10. Select colonies and analyze with PCR or w/e.