Team:Gaston Day School/Notebook

Notebook

On this page, Gaston Day School iGEM team would like to share with you our notes during the experiments from 09/01/2017 to 10/23/2017. The team leader Heena Saqib records the data patiently.

09/01/2017

Materials:
LB broth
Ice
Selection plates

Methods:

1. Thaw 50µL competent E. coli cells on ice for 10 minutes
   
2. Add:
   • 5-10 µl DNA from a ligation reaction mix or
   • 10-100ng DNA of a known plasmid
3. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
4. Place the mixture on ice for 30 minutes. Do not mix.
5. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
6. Place on ice for 5 minutes. Do not mix.
7. Pipette 950 µl of room temperature SOC or LB media into the mixture.
8. Incubate at 37°C and 200-250 rpm for 60 minutes.
9. Mix the cells thoroughly by flicking the tube and inverting.
10. Spread:
    • For ligation reaction DNA: 100µl of each transformation reaction onto a selection plate. For the rest of 900 µL:
      1. Pellet cells at 8000rpm for 3 minutes
      2. Remove and dispense 600 µL of supernatant
      3. Re-suspend cells by light vortexing
      4. Plate resuspended cells as above
    • For known plasmid: 10 & 100 µL of each transformation reaction onto a selection plate. For the rest of 890 µL:
      1. Pellet cells at 8000rpm for 3 minutes
      2. Remove and dispense 600 µL of supernatant
      3. Re-suspend cells by light vortexing
      4. Plate resuspended cells as above
11. Incubate overnight at 37°C with plates upside down.

 

Email: iGEM@gastonday.org

Gaston Day School

2001 Gaston Day School Rd

Gastonia 28056