Transform bacterial DNA through methods using heat shock and recombination
Bacterial (prokaryotic) chromosomes are in the form of hoops and circles and are far smaller than eukaryotic chromosomes. By forcing a plasmid (circular strand of foreign DNA) to enter a bacterial cell, the plasmid can merge into the chromosome can be change the DNA. This process is called transformation. Bacterial cells and the plasmid of interest can be stored in a warm environment (most likely at 37C). A solution can be stored in a Revolutionary Science Incufridge and Poly Pro Bath. During this incubation period, the cell walls become porous and allow the plasmid to enter. Solution can then be transferred to a water bath at 42 degrees Celsius. The abrupt change in temperature will “heat shock” the solution. When the cell is exposed to the elevated temperature, the lipid membrane closes and holds in the plasmid, allowing it to insert itself into the chromosome and reprogram it. Bacterial cells can be inoculated on a petri plate, incubated in an incubator, where colony forming units (CFU’s) can be counted either by hand or with an automated colony counter, like the IncuCount. Aseptic techniques should be employed. Petri plates should be properly disposed of after they are no longer needed, by sterilizing in an autoclave, like the Saniclave 50.
- 1 – 15 mL centrifuge tube containing LB Agar Media powder
- 1 – 15 mL centrifuge tube containing LB Antibiotic Agar Media powder (kanamycin (25 µg/mL), streptomycin (50 µg/mL), and arabinose (1mM))
- 5 – 1.5 mL microcentrifuge tubes containing LB Media powder
- 1 – 1.5 mL microcentrifuge tube containing 1 mL of bacterial cell Transformation Buffer (25mM CaCl2, 10% PEG 8000, and 5% DMSO)
Items are shipped at ambient temperature, but store in freezer upon arrival.
- 1 – Glass vial containing non-pathogenic Escherichia coli HME63 strain bacteria
- 1 – 1.5 mL microcentrifuge tube containing 55 µL of Cas9 and tracrRNA Plasmid (100 ng/µL)
- 1 – 1.5 mL microcentrifuge tube containing 55 µL of crRNA Plasmid (100 ng/µL)
- 1 – 1.5 mL microcentrifuge tube containing 55 µL of Template DNA (100 ng/µL)
Equipment and Instrumentation Not Supplied with Kit
- 1 – Revolutionary Science Incufridge set to 7°C (Recommended, but a refrigerator can be used instead)
- 1 – Revolutionary Science Incufridge set to 37°C (Required)
- 1 – Revolutionary Science Mini Pro Bath set to 99.9°C (Recommended, but a refrigerator can be used instead)
- 1 – Revolutionary Science Poly Pro Bath set to 42°C (Required)
- 1 – Revolutionary Science IncuCount, Automated Colony Counter (Recommended)
Supplies Not Supplied with Kit
- 1 – Graduated cylinder
- 1 – 10-100 µL pipette with appropriate pipette tips
- 1 – 100-1000 µL pipette with appropriate pipette tips
- 1 – 250 mL glass bottle (2 bottles required if making both plate types simultaneously)
- 5 – 1.5 µL microcentrifuge tubes
- 14 – Sterile petri plates with lids
- 15 – Sterile disposable inoculation loops
- Microcentrifuge tube rack
- Water (sterile water is recommended when it is being used as a reagent)
- Heat resistant gloves
- Sterile nitrile gloves
- Safety glasses or goggles
- Lab coat or apron
Always wear sterile nitrile gloves for safety, and to avoid contamination. If gloves become soiled, replace with new gloves immediately.
- Always wear safety glasses or goggles to avoid injury to the eyes.
- Always wear a lab coat or apron to avoid spilling or spraying materials on clothes and skin.
- Always wear heat resistant gloves when handling hot equipment/glassware.
- Treat everything as if it were a potential biohazard.
- Spray and wipe down all work surfaces with 70% isopropyl alcohol before and after performing experimental techniques to maintain a clean environment.
- Discard pipette tip after every use.
- Use bacteria from sufficiently grown LB Agar Media plates as soon as possible. Bacterial plates can be stored up to a week, but using fresh bacteria improves the chances for a successful experiment.
- If experiment is unsuccessful, increase the final incubation time of the competent cell mixture with CRISPR elements in the DNA Transformation and CRISPR section of the Protocol.
Making Agar Media Plates
- Add water to a Revolutionary Science Mini Pro Bath, set temperature to 99.9°C by rotating the dial all the way on high, and turn on. The Poly Pro Bath will be used to heat the agar media solution.
- Pour contents of the 15 mL centrifuge tube labeled “LB Agar Media,” or “LB Antibiotic Agar Media” into a 250 mL glass bottle. Both types of agar media plates need to be made for this experiment. Start with one media type, and repeat this section with the other media type. Both agar media plate types can be made simultaneously if two 250 mL glass bottles are available.
- Add 150 mL of water (sterile water recommended) to the 250 mL glass bottle, and swirl the mixture until all of the agar media powder is thoroughly mixed in the water. Make sure all large clumps of agar are broken up.
- Heat the 250 mL glass bottle containing the agar media solution in the pre-heated Revolutionary Science Poly Pro Bath. If needed, add water to the Poly Pro Bath until the water level is slightly above the agar media solution level in the 250 mL glass bottle. Keep the lid for the 250 mL glass bottle screwed on very loosely to avoid pressure build up.
- When the agar media solution is thoroughly heated, all of the agar media powder should be dissolved, the liquid agar media should be completely clear, and the media should be yellow/light amber in color.
- Turn off Revolutionary Science Poly Pro Bath.
Remove the 250 mL glass bottle from the Poly Pro Bath using heat resistant gloves to avoid being burned.
- Allow the liquid media to cool to 55°C, or until the media is just cool enough to be handled without gloves. Cooling may take up to 30 minutes.
- Gather seven petri plates with lids per media type, and orient them so the lids are facing up. The lids are larger in diameter than the plates.
- One by one, remove the lid from a plate, pour warm liquid media in the center of the plate just enough to achieve full coverage of the bottom, and cover the plate with its lid. Avoid overpouring to conserve enough media to make all seven plates per media type.
- Cool plates at room temperature, or place them inside a Revolutionary Science Incufridge at 7°C for quicker cooling. Cooling will take approximately one hour at room temperature. Plates are sufficiently cooled when the liquid agar media solidifies, and has a gelatin-like consistency. If time permits, let the plates cool for a few hours, or even overnight, to allow condensation to evaporate.
- Once cooled, store the plates upside down in a Revolutionary Science Incufridge at 7°C. Do not allow the plates to freeze.
Making Competent Cells for DNA Transformation
- Obtain the vial containing E. coli bacteria, and a sterile inoculation loop. Gently scrape out enough bacteria to coat the loop.
- With a new LB Agar Media plate (not an LB Antibiotic Agar Media plate), orient the loop so it is parallel with the media surface, and gently spread bacteria using a continuous pattern. If needed, flip loop and continue spreading in order to distribute all visible bacteria to the media surface. Spread bacteria across as much of the media surface as possible, and avoid puncturing the fragile media to allow for optimal bacteria growth.
- Place the lid on the plate immediately after spreading to avoid contamination.
- Invert the plate, and allow bacteria to grow overnight at room temperature, or at 37°C in a Revolutionary Science Incufridge. Allow for more time if growing at room temperature. Bacteria growth is sufficient when off-white bacterial streaks and colonies are visible.
- Once bacteria growth is sufficient, pipette 100 µL of Transformation Buffer into a new 1.5 µL microcentrifuge tube.
- With an inoculation loop, carefully scrape bacteria off of the plate until the loop is coated, and stir the bacteria into the Transformation Buffer. Stir vigorously in order to break up any large clumps of bacteria. If clumps cannot be broken up, carefully pipette mixture up and down. Mixture should be moderately cloudy after bacteria is fully mixed in. If mixture is not cloudy, add more bacteria to the vial.
- Competent cells are ready for immediate use, or can be stored in a Revolutionary Science Incufridge at 7°C for use within 2-3 days.
DNA Transformation and CRISPR
- Add water to a Revolutionary Science Poly Pro Bath, set temperature to 42°C, and turn on. The Poly Pro Bath will be used to heat the competent cell mixture.
- Obtain the microcentrifuge tubes labeled “Cas9 and tracrRNA Plasmid,” “crRNA Plasmid,” and “Template DNA.” Pipette 10 µL from each tube into the competent cell mixture – making sure to use a fresh pipette tip before pipetting from each vial.
- Using a pipette set to 100 µL, pipette competent cell mixture with the CRISPR elements up and down several times to thoroughly mix.
- Incubate the tube containing the competent cell mixture with the CRISPR elements at 7°C in a Revolutionary Science Incufridge for 30 minutes. Do not allow the competent cell mixture to freeze.
- Incubate the same tube at 42°C in the pre-heated Revolutionary Science Poly Pro Bath for 30 seconds.
- Turn off the Revolutionary Science Poly Pro Bath.
- Obtain a microcentrifuge tube containing LB Media powder, and pipette in 1 mL of room temperature water (sterile water recommended). Pipette up and down to dissolve the LB Media powder, and thoroughly mix the solution.
- Pipette 200 µL of LB Media solution into the competent cell mixture with the CRISPR elements.
- Incubate at 37°C in a Revolutionary Science Incufridge for 2 hours, or at room temperature for 4 hours. This step allows the competent cells to undergo the CRISPR process. In order for the CRISPR process to work, do not shorten the time.
- During incubation, take an LB Antibiotic Agar Media plate out of the Revolutionary Science Incufridge at 7°C and let it warm up to room temperature.
- Once incubation is complete, pipette 100 µL of the competent cell mixture with CRISPR elements onto the media surface of the LB Antibiotic Agar Media plate.
- Gently spread the competent cell mixture with CRISPR elements evenly across the plate using an inoculation loop, and let it dry for 5 minutes before putting the lid back on.
- Place the lid on the plate, flip the plate upside down, and incubate at 37°C in a Revolutionary Science Incufridge for 18-24 hours, or at room temperature for 24-48 hours.
- The CRISPR experiment is successful if off-white bacteria colonies grow on the LB Antibiotic Agar Media plate. If no colonies form, repeat experiment, and adjust parameters if necessary.