Revision as of 19:26, 19 June 2017

Overview

Transformations are essential to using the DNA Distribution Kits: resuspend the DNA sample in a well, transform the DNA into competent cells, and select single colonies. However, transformations can also be one of the more fickle laboratory techniques.

At iGEM HQ, we have run test transformations of the DNA Distribution Kit with the following protocols. We have found that these are the best protocols to use with the DNA Distribution Kit to ensure high efficiency transformations. We provide two alternative protocols: Single-tube reactions, for transforming a few samples at a time; and 96-well format transformations, for transforming multiple samples at once. The 96-well format transformation protocol is also appropriate for use in labs where there is no access to a 37°C shaker-incubator.

• At iGEM HQ, we make our own stocks of NEB 10b competent cells. Competent cells purchased from vendors will have better efficiency.
• Make sure to test the competency of your cells with the provided Competent Cell Test Kit.
• Read through the entire protocol before starting!

Single Tube Transformation Protocol

96-Well Transformation Protocol

Printable PDF Protocol
Estimated bench time: 1 hour
Estimated total time: 3 hours (plus 14-18 hour incubation)

Materials

• Resuspended DNA to be transformed
• 10pg/µl Positive transformation control DNA
• Competent Cells (50µl per sample)
• Flat-bottomed 200µL 96-well plate OR 2ml Microtubes
• SOC Media (200µL per sample)
• Petri plates w/ LB agar and antibiotic (2 per sample)

Equipment

• Floating Foam Tube Rack
• Ice & ice bucket
• Lab Timer
• 42°C water bath
• 37°C incubator
• Sterile spreader or glass beads
• Pipettes and Tips (10µl, 20µl, 200µl tips and pipettes recommended)

Setup & Protocol

When transforming competent cells, both timing and temperature are very important. Use a lab timer, follow the incubation temperatures closely, and keep materials on ice when required.

Resuspend DNA in selected wells in the Distribution Kit. Label 2ml tubes with part name or well location. Fill lab ice bucket with ice, and pre-chill 2ml tubes (one tube for each transformation, including your control) in a floating foam tube rack.

1. Thaw competent cells on ice: This may take 10-15min for a 260µl stock. Dispose of unused competent cells. Do not refreeze unused thawed cells, as it will drastically reduce transformation efficiency.
2. Pipette 50µl of competent cells into 2ml tube: 50µl in a 2ml tube per transformation. Tubes should be labeled, pre-chilled, and in a floating tube rack for support. Keep all tubes on ice. Don’t forget a 2ml tube for your control.
3. Pipette 1µl of resuspended DNA into 2ml tube: Pipette from well into appropriately labeled tube. Gently pipette up and down a few times. Keep all tubes on ice.
4. Pipette 1µl of control DNA into 2ml tube: Pipette 1µl of 10pg/µl control into your control transformation. Gently pipette up and down a few times. Keep all tubes on ice.
5. Close 2ml tubes, incubate on ice for 30min: Tubes may be gently agitated/flicked to mix solution, but return to ice immediately.
6. Heat shock tubes at 42°C for 1 min: 2ml tubes should be in a floating foam tube rack. Place in water bath to ensure the bottoms of the tubes are submerged. Timing is critical.
7. Incubate on ice for 5min: Return transformation tubes to ice bucket.
8. Pipette 200µl SOC media to each transformation: SOC should be stored at 4°C, but can be warmed to room temperature before use. Check for contamination.
9. Incubate at 37°C for 2 hours, shaker or rotor recommended:
10. Pipette each transformation on two petri plates for a 20µl and 200µl plating: Pipette 20µl and 200µl of the transformation onto appropriately labeled plates. Spread with sterilized spreader or glass beads immediately. This helps ensure that you will be able to pick out a single colony.
11. Incubate transformations overnight (14-18hr) at 37°C: Incubate the plates upside down (agar side facing up). If incubated for too long, colonies may overgrow and the antibiotics may start to break down; un-transformed cells will begin to grow.
12. Pick single colonies: Pick single colonies from transformations: do a colony PCR to verify part size, make glycerol stocks, grow up cell cultures and miniprep.
13. Count colonies for control transformation: Count colonies on the 20μl control plate and calculate your competent cell efficiency. Competent cells should have an efficiency of 1.5x10^8 to 6x10^8 cfu/µg DNA.

Other Resources

Video

Transforming Your Part from iGEM Videos.

• Please note, this video may be outdated.