Heat shock transformation (E.coli) protocol

Introduction

The heat shock method is one of the most used methods for transforming E.coli with the plasmid of interest.

Applications

• Propagation of plasmid DNA
• Storage of plasmid DNA (in the form of glycerol stock)
• Synthesis of proteins
• To modify the metabolism of bacterial

Reagents

• Competent cells.
• LB or SOC broth media.
• LB agar plates with appropriate selection marker.

Instruments and other requirements

• Incubator shaker.
• Incubator.
• Water bath or heat block.
• Laminar flow hood.
• Centrifuge.
• Pipettes.
• Sterile tips.
• Spreader, and parafilm.

Keep Following ready before starting the transformation

1. Make sure laminar flow is available at the time of spreading (about 1.5-2 hr from the beginning of protocol).
2. Make sure the incubator is available for the entire duration of the experiment and is set for 37°C.
3. Keep the LB agar plates (with appropriate antibiotic) and media (or appropriate media) at room temperature or 37°C.
4. Water bath or heat block at 42°C.

Duration of the experiment

the experiment takes nearly 2 hours to complete the transformation, 12-16 hours after transformation (spreading)for the colonies to appear on the plate.

Procedure

1. Take out the competent cells from -80^◦C and keep them on ice for 5-10 mins.
2. Add the ligation mix or plasmid to the competent cells and mix gently. Do not shake or vortex as it will inhibit transformation efficiency.
3. Incubate the reaction mix on ice for 10-15 minutes.
4. Give a heat shock to the cells by placing the reaction mix at 42°C for 30-90 seconds (water bath or Heat-block).
5. After the heat shock, transfer the cells onto the ice and add 500uL of warm LB
6. Place the tubes in the shaker (180 rpm) at 37°C for 1 hour.
7. After the incubation, give a brief spin at 4000 rpm for 2-3 minutes and decant the supernatant of 400 ul. Mix the pellet with the remaining supernatant (~100uL)
8. Split the mixture into 10uL and 90uL to spread onto different plates with appropriate antibiotics (or auxotrophic selection).
9. Incubate the plates overnight at 37°C.
10. After 12-16 hours of plating, colonies can be observed on the plates.

Variations of the protocol

Step 5:

Adding 250 ul of media to the heat-shocked cells eliminates the spinning step, and one can directly spread the 10 % and 90 % volume on selective plates after 1-hour incubation.

The plating of two different volumes helps in selecting well-spaced individual colonies for the downstream process.

Storage

The agar plates with bacterial colonies can be stored for a week @4°C. However, plates that contain antibiotics with less shelf-life may not be stored as without selection pressure, satellite colonies grow.

Caution

Do not leave the plates for more than the recommended time on incubator as overgrowth of colonies makes it challenging to pick individual ones, and it might produce satellite colonies (without plasmid).