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Team:Imperial College London/Extras/Protocols/Chemotaxis
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<html><h2>28th of July</h2> | <html><h2>28th of July</h2> | ||
Transformation of cells with 6, 7 and 8:<br><br> | Transformation of cells with 6, 7 and 8:<br><br> | ||
| - | -Let competent cell strain 5α thaw for around 10 minutes on ice.<br> | + | - Let competent cell strain 5α thaw for around 10 minutes on ice.<br> |
| - | -Add 2-3μl of DNA.<br> | + | - Add 2-3μl of DNA.<br> |
| - | -Leave on ice for 20-25 minutes.<br> | + | - Leave on ice for 20-25 minutes.<br> |
| - | -Heat shock cells at 42°C for 45 seconds.<br> | + | - Heat shock cells at 42°C for 45 seconds.<br> |
| - | -Leave on ice for 10 minutes.<br> | + | - Leave on ice for 10 minutes.<br> |
| - | -Add 500μl of LB broth.<br> | + | - Add 500μl of LB broth.<br> |
| - | -Incubate for 1 hour at 37°C.<br> | + | - Incubate for 1 hour at 37°C.<br> |
| - | -Centrifuge for 1 minute.<br> | + | - Centrifuge for 1 minute.<br> |
| - | -Remove 100μl off the top of the eppendorf tube. Pour out the rest making sure that the pellet remains in the eppendorf tube.<br> | + | - Remove 100μl off the top of the eppendorf tube. Pour out the rest making sure that the pellet remains in the eppendorf tube.<br> |
| - | -Re-suspend the cells in the 100μl LB broth solution that was removed in the previous step. | + | - 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).<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> | <html><br><br> | ||
To make tryptone broth (bacterial growth before chemotaxis assays)for total volume of 1L:<br> | To make tryptone broth (bacterial growth before chemotaxis assays)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> |
| - | -1.14g kanamycin<br> | + | - 1.14g kanamycin<br> |
<br> | <br> | ||
To make 1X PBS (phosphate buffer saline):<br> | To make 1X PBS (phosphate buffer saline):<br> | ||
-in 800ml of distilled H<sub>2</sub>O<br> | -in 800ml of distilled H<sub>2</sub>O<br> | ||
| - | -8g of NaCl<br> | + | - 8g of NaCl<br> |
| - | -0.2g of KCl<br> | + | - 0.2g of KCl<br> |
| - | -1.44g of Na<sub>2</sub>HPO<sub>4</sub><br> | + | - 1.44g of Na<sub>2</sub>HPO<sub>4</sub><br> |
| - | -0.24g of KH<sub>2</sub>PO<sub>4</sub><br> | + | - 0.24g of KH<sub>2</sub>PO<sub>4</sub><br> |
| - | -adjust pH to 7.4<br> | + | - adjust pH to 7.4<br> |
| - | -adjust volume 1L with additional distilled H<sub>2</sub>O<br> | + | - adjust volume 1L with additional distilled H<sub>2</sub>O<br> |
| - | -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> | ||
| + | <h2>2nd of August</h2> | ||
| + | Preparation of semi-solid agar used for qualitative experiments, the recipe is for total volume of 1 litre <br> | ||
| + | - 5g NaCl<br> | ||
| + | - 10g tryptone<br> | ||
| + | - 2g D-glucose<br> | ||
| + | - 3g agar<br> | ||
| + | - 1000ml H<sub>2</sub>O<br> | ||
| + | - autoclave<br> | ||
| + | - before making plates, cool down semi-solid agar to 50<sup>o</sup>C and add required amount of antibiotics | ||
</html> | </html> | ||
Revision as of 21:20, 3 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.
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 50oC and add required amount of antibiotics
</html>
