Mix & Go Chemically Competent E. coli

Writing/editing credits: Tiffany Lowe-Power,

Spreadsheet of Competent E. coli (dates prepped, genotypes, and transformation efficiencies)

We use a modification of the the Zymo Mix & Go Transformation Kit protocol. Make a large batch. Cells should be “good” for > 1 yr if stored at -80C.

Protocol

This protocol takes 4 days if desired strain of E. coli must be streaked from the -80C freezer. The most time intensive step is the morning of the fourth day. Briefly:

1. Streak desired E. coli strain from -80C freezer.
2. Pick a single colony into a 2-5 mL overnight culture.
3. Use 2-5 mL culture to start a larger overnight culture in a flask.
4. Harvest cells from flask at mid-log phase and prepare + save competent cells.

Prep Checklist

• Zymo Mix & Go buffers (2x stocks). You need > 2.5 ml of each. These are stored in the 4C fridge, and must be used ice-cold.
• Sterile flask (125 ml - 250 ml)
• Room temperature shaker incubator (usually stored in a cabinet)
• Tubes for competent cell aliquots (1.5 ml is best). Prep 60 per batch of cells you are making. Label with a brief name like “5α”. Leave most of the lid open so that users can write their plasmid on the lid. Keep these at -20 until ready to use.
• Pre-label a freezer box. e.g. “Mix & Go E. coli 5-alpha // Prepped 2020-12-31 by TLP”

Steps

Perform these steps in the laminar flow hood or the biological safety cabinet.

1. Streak E. coli 5-alpha onto an LB plate (no antibiotics!). Grow overnight at 37 C. Can be stored at 4C for up to 1-2 months if wrapped with parafilm to keep agar hydrated.
2. Inoculate a single colony of E. coli 5-alpha in 2-5 ml LB. Incubate with shaking: 37C overnight.
3. At 5pm the next day, subculture the overnight culture of E. coli 1:100 (e.g. 500 ul into 50 ml broth). You can use Zymobroth or LB.
4. Incubate at Room temperature overnight with shaking at 150 rpm (we keep the RT shaker in a cabinet).
5. At 8-9 am the next morning, check the OD of the culture (do a 1:10 dilution to minimize how many cells you waste). You want to catch the E. coli at OD 0.4 - O.6 for the highest efficiency cells.
   • As E. coli approaches stationary phase, they will become less transformable. The Zymo protocol links an excellent paper that show ideal ODs and incubation temperatures for highly competent E. coli https://pubmed.ncbi.nlm.nih.gov/2265755/
6. When culture is at the appropriate OD (0.4 - O.6), chill the flasks on ice (make sure the flask is slightly buried so that the full culture volume chills). Chill on ice for at least 10 minutes. While culture is chilling:
   • Cool the 50 ml centrifuge to 4 C.
   • Prepare 1x stocks of the Wash buffer and the Competent buffer. These buffers must be kept ice-cold–keep in ice. The 1x solutions expire after 48 hrs. The volumes below are sufficient for 1 batch of cells. Scale up as needed.
   • Mix 2.5 ml of the Dilution buffer with the 2x Wash buffer to make 1x Wash buffer. Store in ice.
   • Mix 2.5 ml of the Dilution buffer with the 2x Competent buffer to make 1x Competent buffer. Store in ice.
7. After 10 minutes on ice, pellet the cells by centrifugation at 2,500 x g (rcf) for 10 minutes at 0 - 4°C.
8. Remove the supernatant and resuspend the cells gently in 5 ml ice-cold 1X Wash Buffer.
9. Pellet the cells by centrifugation at 2,500 x g (rcf) for 10 minutes at 0 - 4°C.
10. Completely remove the supernatant. (If necessary, briefly spin the cells again. Use a P1000 to remove all of the supernatant).
11. Gently resuspend the cells in 5 ml ice-cold 1X Competent Buffer. (Using 10 ml pipettes + pipetted gun works well)
12. Quickly move all of the labeled 1.5 ml tubes from the freezer to the ice bucket.
13. Aliquot (on ice) 100 ul of the cell suspension into sterile microcentrifuge tubes. Cells are now ready for transformation with DNA or can be stored below -70°C for transformation at a later time.
    • Store in Rack 16 with the E. coli strains (275 freezer)
    • Add information into the Spreadsheet of Competent E. coli (dates prepped, genotypes, and transformation efficiencies)
14. Check the transformation efficiency of the cells using pUC19 (AmpR plasmid that comes with the NEB Assembly master mix). Record transformation efficiency into the spreadsheet.

Resources

Add Gene’s advice

Controls with each batch:

• Negative Control: Plate residual cells on antibiotic plates to ensure there’s no contamination.
• Positive Control: Transformation with xxx ng of xxx plasmid
• First person to make in Lowe-Power lab – create a lab & link to spreadsheet to record parameters… # tubes created, whether cells passed negative control, & # colonies. Also, flesh out a brief protocol for the + control experiment.

Important notes:

• Growth temperature: Generally the cooler the growth temperature, the slower the E. coli grow, which dramatically improves competence.
  • Lowe-Power lab has a room-temperature shaker incubator that should be used for competent E. coli.
  • In the Cook lab, ambient temperature around 22 degrees C, Stratton saw 20 mL of zymobroth inoculated with 0.5 mL overnight culture grow to an OD600 of 0.792 in 21.5 hours.
• Growth medium … to be determined in our own hands. We have ZymoBroth. First person in the lab gets to compare that to LB & update this protocol with a note about (1) relative efficiency of the resulting cells (2) how long it took cells to reach the proper OD for harvesting. Thanks!