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Procedure
- Thaw competent cells on ice for about 45 min (use approximately 120 μl in 1.5 ml eppendorf).
- Add ligation mixture (or appropriate positive or negative control) – approximately 10-15 μl.
- Flick the bottom of the eppendorf gently to mix.
- Incubate on ice for 30 min.
- Heat “shock” the cells for transformation by placing in 42°C bath or heating plate for 30 sec only.
- Place cells back on ice.
- Add 500 μl of LB media
- Shake in an orbital shaker at approx 240 rpm for 90 min.
- Centrifuge the solution at 12,000 rpm for 30 sec to pellet the bacteria.
- Pour off most of the supernatant leaving behind approximately 50 μl of media above the pelleted bacteria.
- Re-suspend the bacteria into the remaining LB and then plate onto LB/Aapicillin plates (or LB agar plates with the appropriate antibiotic) and incubate overnight at 37°C.
- The next morning, check the plate(s) for colonies. Scrape a portion of individual colonies with a sterile pipet tip or toothpick and immerse in a sterile culture tube containing LB media (3 ml) supplemented with ampicillin (6 μl of 5% ampicillin stock). Shake in an orbital shaker at 37°C for at least 2 hours.
- When the solution has reached a reasonable level of cloudiness from bacterial growth, use approximately half of the bacterial culture (1.5 ml) for a mini-prep and perform a restriction digest on the resultant plasmid that’s isolated. If the appropriate insert is seen on Agarose gel electrophoresis, you can check the remaining DNA by sequencing.
- For plasmid DNA preparation, place approximately 1 ml of the 3 ml culture from above into a sterile flask (500 ml) containing approximately 150-200 ml of LB media plus ampicillin (400 μl of 5% stock into 200 ml of LB). Shake in an orbital shaker overnight at 37°C. On the next day, continue with DNA preparation.