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From 2011.igem.org

Chemotaxis Lab Protocols

27th of July

The strain of E. coli with the copy of super-folded GFP that was incubated the previous night has shown limited/no growth in the prepared LB broth. This could have been caused by:
-No innoculation
-Kanamycin concentration in LB broth was too high. We had used 85μg/ml.

New protocol:
50μg/ml is the recommended concentration. We will use 35-40μg/ml of kanamycin to speed up growth.

28th of July

Transformation of cells with 6, 7 and 8:

- Let competent cell strain 5α thaw for around 10 minutes on ice.
- Add 2-3μl of DNA.
- Leave on ice for 20-25 minutes.
- Heat shock cells at 42°C for 45 seconds.
- Leave on ice for 10 minutes.
- Add 500μl of LB broth.
- Incubate for 1 hour at 37°C.
- Centrifuge for 1 minute.
- Remove 100μl off the top of the eppendorf tube. Pour out the rest making sure that the pellet remains in the eppendorf tube.
- Re-suspend the cells in the 100μl LB broth solution that was removed in the previous step. - Add 5μl on a chloramphenicol agar plate (concentration of 35μg/ml).
- Add the rest of the sample to a second chloramphenicol agar plate.


To make tryptone broth (bacterial growth before chemotaxis assays)for total volume of 1L:
- 10g tryptone
- 1000ml of 1X PBS
- autoclave
- 1.14g kanamycin

To make 1X PBS (phosphate buffer saline):
-in 800ml of distilled H₂O
- 8g of NaCl
- 0.2g of KCl
- 1.44g of Na₂HPO₄
- 0.24g of KH₂PO₄
- adjust pH to 7.4
- adjust volume 1L with additional distilled H₂O
- autoclave
Note: also possibility to use 1X PBS tablets (one tablet per 200ml)

2nd of August

Preparation of semi-solid agar used for qualitative experiments, the recipe is for total volume of 1 litre
- 5g NaCl
- 10g tryptone
- 2g D-glucose
- 3g agar
- 1000ml H₂O
- autoclave
- before making plates, cool down semi-solid agar to 50^oC and add required amount of antibiotics </html>