Team:Aachen/Wetlab/Protocol14

E. coli Tingle Tube Transformation for Interlab Study:

Before starting:

Estimated bench time: 1 hour. Estimated total time: 2 hours (plus 14-18 hour incubation). 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. Read through the entire protocol before starting!

Material:


  • Resuspended DNA to be transformed.

  • 10 pg/ul Positive transformation control DNA (e.g. pSB1C3 w/ BBa_J04450, RFP on high-copy chloramphenicol resistant plasmid. Located in the *Competent Cell Test Kit.)

  • competent cells (50 ul per sample).

  • 1.5 mL Microtubes.

  • SOC Medium (950 uL per sample)

  • LB Agar + Chloramphenicol (2 per sample.)

Equipment:


  • Floating Foam Tube Rack.

  • Ice & ice bucket.

  • Lab Timer.

  • 42 ℃ water bath.

  • 37 ℃ incubator

  • Sterile spreader or glass beads (/ also "dreigalsky-spatel" works)

  • Pipettes and Tips (10 ul, 20 ul, 200 ul recommended)

  • Microcentrifuge

Equipment:


  1. Resuspend DNA in selected wells in the Distribution Kit with 10 ul dH20. Pipet up and down several times, let sit for a few minutes. Resuspension will be red from cresol red dye.

  2. Label 1.5 ml tubes with part name or well location. Fill lab ice bucket with ice, and pre-chill 1.5ml tubes (one tube for each transformation, including your control) in a floating foam tube rack.

  3. Thaw competent cells on ice: This may take 10-15 min for a 260 ul stock. Dispose of unused competent cells. Do not refreeze unused thawed cells, as it will drastically reduce transformation efficiency.

  4. Pipette 50 ul of competent cells into 1.5 ml tube: 50 ul in a 1.5 ml tube per transformation. Tubes should be labeled, pre-chilled, and in a floating tube rack for support. Keep all tubes on ice. Do not forget a 1.5 ml tube for your control.

  5. Pipette 1µl of resuspended DNA into 1.5 ml tube: Pipette from well into appropriately labeled tube. Gently pipette up and down a few times. Keep all tubes on ice.

  6. Pipette 1 ul of control DNA into 2 ml tube: Pipette 1 ul of 10 pg/ul control into your control transformation. Gently pipette up and down a few times. Keep all tubes on ice.

  7. Close 1.5 ml tubes, incubate on ice for 30min: Tubes may be gently agitated/flicked to mix solution, but return to ice immediately.

  8. Heat shock tubes at 42 ℃ for 45 sec: 1.5 ml 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.

  9. Incubate on ice for 5 min: Return transformation tubes to ice bucket.

  10. Pipette 950 ul SOC media to each transformation: SOC should be stored at 4 ℃, but can be warmed to room temperature before use. Check for contamination.

  11. Incubate at 37 ℃ for 1 hours, shaking at 200-300 rpm.

  12. Pipette 100 uL of each transformation onto petri plates Spread with sterilized spreader or glass beads immediately(/"Dreigalsky-Spatel"). This helps ensure that you will be able to pick out a single colony..

  13. Spin down cells at 6800 g for 3 mins and discard 800 uL of the supernatant. Resuspend the cells in the remaining 100 uL, and pipette each transformation onto petri plates Spread with sterilized spreader or glass beads immediately. This increases the chance of getting colonies from lower concentration DNA samples.

  14. Pipette 1 ul 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.

  15. Incubate transformations overnight (14-18 h) at 37 ℃: Incubate the plates upside down (agar side up). If incubated for too long, colonies may overgrow and the antibiotics may start to break down; un-transformed cells will begin to grow.

  16. 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

  17. Count colonies for control transformation: Count colonies on the 100 ul control plate and calculate your competent cell efficiency. Competent cells should have an efficiency of 1.5x10^8 to 6x10^8 cfu/ug DNA.