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===Transformation of E. coli=== *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/ligated cloning vector from above 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.
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