- Goal: To successfully have competent E. coli take in naked DNA and its subsequent stable maintenance.
First, answer these questions:
- Which type of LB plate is required?
Refer to this link for general guidance on making media: http://microbes.sites.haverford.edu/LaboratoryWiki/Media_Recipes
- Which traits do we want in the transformants, especially with regards to having an intact lacZ gene?
- Which cells will these be put into? (traditionally, DH5alpha for plasmid work and BL21 for protein expression)
- Do we have a dilution of the positive control DNA? If not, use the DNA strain database to find the concentration of DNA in the stock of your plasmid of interest and use that to calculate a dilution to give you 25ngDNA/10µL (equal to 2.5ng DNA/µL).
Protocol (Version 3 / Feb 2020):
- Create enough LB plates with the appropriate antibiotic, as well as IPTG and X-gal if you are using blue/white screening.
- Turn on small water bath to 42ºC.
- Turn on the large shaking incubator to 37ºC (or keep at 34ºC, if that is what it already is set at).
- Spray a beaker with ethanol, go get ice from the second floor imaging room (across from lab).
- From the -80 freezer: get one tube of competent cells for each transformation, plus one for a positive control (intact plasmid only), plus one for a negative control (water only). Each freezer tube has 50uL cells. Place tubes into the ice.
- Wait 10-15 minutes for these cells to thaw, always on ice.
- Add ~25ng of ligated plasmid DNA to one tube of cells, and repeat for each transformation. Also add 25ng of empty vector to a separate tube of cells. Add 10uL of sterile water to the negative control tubes. Place back on ice. As you add these small volumes, mix gently with tip of your pipette.
- Allow the cells to incubate for 15 minutes on ice. Do not mix the tubes during this incubation period.
- After 15 mins, carry the ice beaker to the water bath pre-heated to 42ºC. Heat shock the cells for exactly 45 seconds, then immediately return the tubes to ice for 2 minutes.
- Add 250 ul of room temperature LB to each of your tubes.
- Incubate the tubes in the large floor shaking incubator set to 34°C/37ºC, on their side. Make sure tubes are completely shut.
- After 30 minutes, plate cells. Divide ligated plasmids that are going into DH5alpha cells into two plates of 50µL and the rest of the volume (260µL). The entire volume can be plated onto one plate for Gibbon Assembly plasmids and for BL21 cells.
- Spread plates with sterile beads. Put lid on plate and flip over so beads fall onto lid. Pour used beads into white container with funnel (by the sink). Sterilize this funnel with 70% ethanol after use.
- Incubate overnight. You can use the 37°C incubator with ambient CO2, located near the weigh station (only if you are transforming a nonpathogenic bacteria).
Note: If you are using Kanamycin plates, a large number small colonies will appear after 48 to 72 hours. These are not transformed colonies. We are not sure why this happens. Feel free to figure it out.