E coli Heat Shock Transformation Protocol

BBD/JBEI XL1, S17-1 Chemically Competent Cells

Prepared by QB3/Macrolab at UC Berkeley

Transformation efficiency: 4 x 10⁷ cfu/µg pUC19 DNA

This protocol was written by Thomas Eng and adapted from notes from Chris Jeans (UC Berkeley, qb3 Macrolab)

1. Take a single use aliquot from the -80C freezer

2. Thaw aliquot on ice (10 minutes). When most of the ice has melted, add 1-10µL of plasmid DNA, adding no more than 100ng of DNA. Do not vortex.

3. Incubate competent cells + DNA on ice for exactly 30 minutes.

4. Heat shock aliquots in a water bath at 42˚C for 60 seconds.

5. Return cells to incubate on ice for 5 minutes.

6. Add 250µL SOC media (LB is fine, but SOC is better). No antibiotics at this step.

7. Incubate at 37˚C for at least 1 hour with shaking (use a benchtop variable temperature mixer set to 37C – don’t tape your tubes to a Kuhner.)

8. Spread the entire transformation reaction on prewarmed selective plates with glass beads or a sterile glass rod. (Obviously if this is plasmid from a miniprep and not a cloning reaction, you don’t need to plate the entire thing.) Allow plates to dry before inverting, and incubate at 37˚C overnight. Use a minimum number of strokes with the glass rod to spread cells. Look for the liquid media to form rivers on the plate. If you use glass beads, make sure you don’t shake the plates so hard that the glass beads simply spin along the sides of the petri dish. You are aiming to spread the culture, not to dry the plate. Excess spreading can lyse cells!! After the culture media is spread dry the plates in the tissue culture hood or next to an open flame.

9. Transformants should appear the next morning.

TE Note: This protocol can also be used for commercially prepared DH10B cells from NEB with one modification. In step 4, change the heat shock time from 60 seconds to exactly 30 seconds.