Team:Duke/Notebook/Protocols

Chemically Competent Cells

This protocol for making chemically competent E. coli for transformations. It is derived from the official iGEM protocol, which can be found here

1. Making CCMB 80 Buffer
   • 10 mM KOAc pH 7.0 (10 ml of a 1M stock/L)
   • 80 mM CaCl2.2H2O (11.8 g/L)
   • 20 mM MnCl2.4H2O (4.0 g/L)
   • 10 mM MgCl2.6H2O (2.0 g/L)
   • 10% glycerol (100 ml/L)
   • adjust pH DOWN to 6.4 with 0.1M HCl if necessary
2. Culturing Cells
   • Scrape cells from a colony or frozen stock of the desired strain
   • Inoculate into 5 mL SOC (or LB+Antibiotic for plasmid-containing strains)
   • Grow for ~8 hrs (morning to late afternoon) in 37C shaker
   • Add 5 mL of culture to 250 mL SOC (or LB+Antibiotic) in a shaker flask
     • 250 mL culture will yield approximately 50 chemically competent samples. For smaller batches, we add 1 mL into 50 mL.
   • Grow overnight for ~16 hrs in a shaker at room temperature
3. Treating cells
• Transfer culture into 50 mL centrifuge tubes
• Pellet cells at 4500 RPM for 10 mins
• Pour off supernatant and resuspend cells in 40 mL CCMB 80 Buffer
• Incubate on ice for 20 minutes
• Pellet cells at 4500 RPM for 10 mins
• Pour off supernatant and resuspend cells in 5 mL CCMB 80 Buffer
• Incubate on ice for 20 minutes
• Aliquot 750 uL each into pre-chilled microcentrifuge tubes
• Store at -80C until use
• Note: we never refreeze competent cells once thawed. Any unused cells in an aliquot are discarded.

Transformation

This is our standard heat-shock transformation protocol for E. coli cells. We use it for ligations and other assemblies as well as transformation of intact plasmids (i.e. stocks from the distribution kit).

1. Thaw chemically competent cells on ice
2. Add 50 uL cells to 10 uL DNA (plasmid or assembly reaction)

Ligations

We had some trouble with ligations earlier in the year, and our protocol has undergone changes in an attempt to improve our ligation success. This protocol represents the most recent (and most successful) version of our ligations.

1. Restriction digestion
   • To insert a single part into a desired backbone, digest both the insert and backbone with EcoRI and SpeI
   • To insert a new part downstream of an existing part, digest the backbone with PstI and XbaI, and the insert with PstI and SpeI
   • Digest four individual tubes of each mini prepped plasmid
   • Add 10 uL Cutsmart buffer and 5 uL total enzyme, along with dH20 to a final volume of 100 uL for each mini prep
   • Incubate at 37C for 3+ hrs
2. Gel extraction
• Load 200 uL (two digestion tubes) plus 20 uL loading dye into each lane of a 0.8% agarose/TAE gel
• Run gel electrophoresis at 100 volts for 18 minutes
• Cut desired band from gel
• Extract DNA from gel band using Zymoclean gel DNA recovery kit
3. Antarctic Phosphatase treatment
   • Treat only the backbone with phosphatase
   • We used Calf Intestinal Phosphatase earlier in the year, but seemed to have better results with Antarctic Phosphatase
   • Combine two samples of gel extracted DNA (60 uL) with 10 uL Antarctic Phosphatase buffer, 2.5 uL phosphatase, and dH20 to a final volume of 100 uL
   • Incubate for 1-2 hours at 37C
   • Clean up sample by running our PCR cleanup protocol using the Qiagen miniprep kit
   • Typical yield from minipreps to cleaned, processed DNA is about 10-25%, which is why we start with 4 minipreps to obtain one tube of product.
4. Ligation
   • Combine the following in a 0.5 mL microcentrifuge tube:
     • 100 ng backbone DNA
     • 3x molar excess of insert DNA (ex. 150 ng for an insert half the size of the backbone)
     • 1 uL T4 Ligase buffer
     • 0.5 uL T4 Ligase
   • Incubate for 1 hour at room temperature (shorter incubation times do not seem to affect efficiency, but this was our default)
   • Transform into chemically competent E. coli

Protocol

This is a standard protocol for achieving this result.

1. Step 1
2. Step 2
3. Step 3

Protocol

This is a standard protocol for achieving this result.

1. Step 1
2. Step 2
3. Step 3