Prerequisite Protocols

• Transformed bacterial colonies (works with any microbe) already grown on solid media in a petri dish

Materials

• Colony Cracking Buffer 2x
• PCR Tubes
• LB Broth (Miller Mod) + Antibiotics (same as colony plate)
• 2mL Round Bottom Tubes
• Agarose
• TEA 1x Running Buffer

Equipment

• Vortexer
• Incubator Shaker or Tube Thermoshaker
• Gel Electrophoresis Rig

Step By Step

1. Determine how many colonies you wish to crack and label that many 2mL round bottom tubes. Label one additional tube as a negative control.
2. Add 200uL of LB Broth (Miller Mod) + appropriate antibiotics (same as petri dish media) to each tube.
3. Using a 200uL pipette tip or sterile tooth pick, select well isolated colonies and place them, one per 2mL tube. Pipette up and down if using a pipette tip to fully resuspend the lifted colony cells off the tip and into the broth. Do not add cells to the negative control tube.
4. Incubate the cells at 37C for 4hrs shaking in a thermoshaker set to 1000rpm (2mm orbit) or let them rattle around in a box inside an incubator shaker set to 250rpm (20mm+ orbit). If using a box, make sure to secure the box so it does not dance around in your shaker. Set a timer for 3 hours, we will use this in the next step.
5. With one hour remaining on the time (when the 3hr timer goes off), pour an agarose gel with enough wells to fit all of your colony tubes + DNA ladder. Let this set while we wait for the total 4hr incubation to complete.
6. To as many 0.2mL PCR tubes as you have colonies to analyze, add 10uL of Colony Cracking Buffer 2x to each tube. If you have 8 or more colonies to analyze, consider using PCR Tube Strips.