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Checking the colonies
- Use either 10-50 ng of DNA in a volume between 10 and 25 μL or entire ligation reaction and place the tube on ice.
- Thaw 100 μL of competent E. coli by warming it between hands. Place on ice to avoid over-heating the E.coli. Note: When taking the E. coli out of the freezer, check to make sure there is no liquid in the microcentrifuge tube. If liquid is present in the tube, throw it out, as it is most likely ethanol, which would have a negative impact on the transformation results.
- Add DNA to 100 μL of competent E. coli in a microcentrifuge tube by flame.
- Mix gently by tapping the side of the tube.
- Place tube on ice for 10 minutes.
- Heat shock cells by incubating the tube at 42°C for 1:30-2 minutes.
- Place tube on ice for 1-2 minutes.
- Add 1 mL of autoclaved SOC medium for optimal results. Alternately, LB broth may be used. Note: SOC medium recipe/LB broth recipe,
- Shake tube for 1 hour at 37°C, shaking at 800 rpm. While tubes are shaking, get out the antibiotic plates that will be used and allow them to sit at room temperature. Also, if pBluescript is being used, add 100 μL of EZ-GAL to the ampcillin plate once it has warmed up to room temperature and spread it.
- Plate aliquots of the transformation to LB plates containing the appropriate antibiotic. Note: to plate smaller volumes, centrifuge the transformation at 4,500 xg for 1 minute, discard the supernatant, and resuspend the pellet in 20-100 μL of LB medium.
- Incubate plate(s) overnight at 37°C.
Checking the colonies
- Pick colonies and add to 6 mL of LB and 6 μL of the antibiotic in a 15 mL tube.
- Add the tube(s) to shake in an incubator overnight at 37°C.
- In the morning, remove 1 mL of the culture and put it in a microcentrifuge tube.
- Centrifuge the tube at 4,500 xg for 1 minute.
- Discard the supernatant, and resuspend the pellet in 1 mL of LB + 20% glycerol.
- Place in -20°C freezer until sequence has been verified.
- Conduct a mini prep on the remaining 5 mL culture using Geneaid mini prep kit. Note: use 30 μL of ddH2O to elute DNA.
- PCR a total volume of 10 μL for each sample, using a trace amount of DNA (~0.2-0.3 μL)
- Run a gel. Any sample that shows a band with the correct amount of base pairs should be sent to sequencing.