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Team:Imperial College London/Extras/Protocols/Chemotaxis
From 2011.igem.org
(Difference between revisions)
| Line 21: | Line 21: | ||
- Add 5μl on a chloramphenicol agar plate (concentration of 35μg/ml).<br> | - Add 5μl on a chloramphenicol agar plate (concentration of 35μg/ml).<br> | ||
- Add the rest of the sample to a second chloramphenicol agar plate.<br></html> | - Add the rest of the sample to a second chloramphenicol agar plate.<br></html> | ||
| - | <html><br><br> | + | |
| - | To make tryptone broth | + | <html><br> |
| + | <b>Antibiotics</b>:<br> | ||
| + | Four different antibiotics (kanamycin, chloramphenicol, ampicillin & tetracycline) have been used during the course of the project. They have been used in following working concentrations, unless stated otherwise:<br> | ||
| + | - Kanamycin - 35µg/ml<br> | ||
| + | - Chloramphenicol – 35µg/ml<br> | ||
| + | - Tetracycline – 35 µg/ml<br> | ||
| + | - Ampicillin – 100 µg/ml<br><br> | ||
| + | <b>Tryptone broth</b><br> | ||
| + | To make bacteria develop flagella they are grown in the tryptone broth instead of LB broth. This is recipe for total volume of 1L:<br> | ||
- 10g tryptone<br> | - 10g tryptone<br> | ||
- 1000ml of 1X PBS<br> | - 1000ml of 1X PBS<br> | ||
- autoclave<br> | - autoclave<br> | ||
| - | - | + | - add required amount of antibiotics<br> |
<br> | <br> | ||
To make 1X PBS (phosphate buffer saline):<br> | To make 1X PBS (phosphate buffer saline):<br> | ||
| Line 38: | Line 46: | ||
- autoclave<br> | - autoclave<br> | ||
Note: also possibility to use 1X PBS tablets (one tablet per 200ml)<br> | Note: also possibility to use 1X PBS tablets (one tablet per 200ml)<br> | ||
| - | </html> | + | </html><html> |
| - | <h2>2nd of August</h2> | + | <h2>2nd of August</h2><br> |
| - | + | <b>Semi - solid agar</b><br> | |
| + | In chemotaxis assays semi-solid agar is used as it allows greater diffusion of molecules and allows movement of bacteria within agar. This is recipe for total volume of 1 litre: <br> | ||
- 5g NaCl<br> | - 5g NaCl<br> | ||
- 10g tryptone<br> | - 10g tryptone<br> | ||
| Line 47: | Line 56: | ||
- 1000ml H<sub>2</sub>O<br> | - 1000ml H<sub>2</sub>O<br> | ||
- autoclave<br> | - autoclave<br> | ||
| - | - before making plates, cool down semi-solid agar to 50<sup>o</sup>C and add required amount of antibiotics | + | - before making plates, cool down semi-solid agar to 50<sup>o</sup>C and add required amount of antibiotics<br> |
| + | <h2>5th of August</h2> | ||
| + | |||
</html> | </html> | ||
Revision as of 17:25, 7 August 2011
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.
Antibiotics:
Four different antibiotics (kanamycin, chloramphenicol, ampicillin & tetracycline) have been used during the course of the project. They have been used in following working concentrations, unless stated otherwise:
- Kanamycin - 35µg/ml
- Chloramphenicol – 35µg/ml
- Tetracycline – 35 µg/ml
- Ampicillin – 100 µg/ml
Tryptone broth
To make bacteria develop flagella they are grown in the tryptone broth instead of LB broth. This is recipe for total volume of 1L:
- 10g tryptone
- 1000ml of 1X PBS
- autoclave
- add required amount of antibiotics
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
Semi - solid agar
In chemotaxis assays semi-solid agar is used as it allows greater diffusion of molecules and allows movement of bacteria within agar. This is recipe 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 50oC and add required amount of antibiotics
