— Jacob Anderson 2018/11/16 13:29

This protocol is adapted from Kristan Steffen, (UW). This protocol is meant to make more plasmids! Through exogenous uptake we can introduce new constructs to bacteria, force their machinery to make it copies of the desired plasmid, and harvest the product. The use of chemically competent E.coli cells allow for easy transformations while maintaining the plasmid without rearrangement of the plasmid DNA.

• Shaking incubator at 37 °C

• Heat bath at 42 °C

• Ice bucket filled with ice

• Microcentrifuge tubes

• Sterile spreading device “Glass Hockey Stick”

• LB agar plate (with appropriate antibiotic)

• LB or SOC media

• Competent E. coli cells

• DNA you'd like to transform

-1. Make sure that the 42°C heating block is turned on and heating up.

0. Remove the competent E. coli from -80C freezer and allow to thaw on ice.

1. Aliquot 50 uL of chemically competent E. coli in individual 1.5ml flip top eppie tubes. The box may read “Chemically Competent E. Coli” or simply C2 or C3. “Other E.coli strains include DH5alpha.”

1. Add 0.5 - 1.0 uL plasmid into the 1.5 mL flip tube. GENTLY mix by flicking the bottom of the tube with your finger a few times. 2. Incubate the competent cell/Plasmid mixture on ice for 20-30 mins

3. Heat shock: 45 seconds @ 42°C heat bath.

4. Put it back on the ice.

5. Add 100 uL LB. Incubate @ 37°C between 5 minutes to 5 hours. Currently, we use the 37C shaker in CRF on the first floor.

6. Pipette your E.coli/plamsid mixture onto LB media that includes your choice of drug marker (Drug, -URA, etc) your plasmid has for bacteria. leave overnight in the 37 °C incubator and check for colonies the next day. Again, we use, currently, the first floor shaker in the CRF.