Team:UCL London/NZY+ Broth (per Liter)

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==NZY+ Broth (per Liter)==
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=='''NZY + Broth'''==
 +
''(per litre)''
 +
 +
  10 g of NZ amine (casein hydrolysate)
 +
 +
  5 g of yeast extract
 +
 +
  5 g of NaCl
-
10 g of NZ amine (casein hydrolysate)
 
-
5 g of yeast extract
 
-
5 g of NaCl
 
Add deionized H2O to a final volume of 1 liter.  Adjust to pH 7.5 using NaOH.
Add deionized H2O to a final volume of 1 liter.  Adjust to pH 7.5 using NaOH.
 +
Autoclave.
Autoclave.
 +
Add the following filer-sterilized supplements prior to use:
Add the following filer-sterilized supplements prior to use:
 +
12.5 ml of 1 M MgCl2
12.5 ml of 1 M MgCl2
 +
12.5 ml of 1 M MgSO4
12.5 ml of 1 M MgSO4
 +
20 ml of 20% (w/v) glucose (or 10 ml of 2 M glucose) Transformation:
20 ml of 20% (w/v) glucose (or 10 ml of 2 M glucose) Transformation:
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Materials:
 
-
Plasmids DNA
 
-
Competent E.coli
 
-
NZY Medium
 
-
LB buffer
 
-
Ampicllin, Kanamycin, Tetracycline
 
-
Agar Plates
 
-
Eppendorf
 
-
'''Method:'''
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=== Material ===
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  Plasmids DNA
 +
 +
  Competent E.coli
 +
 +
  NZY Medium
 +
 +
  LB buffer
 +
 +
  Ampicllin, Kanamycin, Tetracycline
 +
 +
  Agar Plates
 +
 +
  Eppendorf
 +
 +
 +
==='''Method'''===
 +
[[Image:UCL-Plate1.JPG |400px|right]]
1. Punch the appropriate well with a clean tip.
1. Punch the appropriate well with a clean tip.
 +
2. Soak the spots in 10µL of TE that has been warmed to 50ºC for 20 minutes. At this time take the competent cells from the -80º freezer and start defrosting on ice.
2. Soak the spots in 10µL of TE that has been warmed to 50ºC for 20 minutes. At this time take the competent cells from the -80º freezer and start defrosting on ice.
 +
3. Add 4µL of DNA in TE to 100µL of competent cells.
3. Add 4µL of DNA in TE to 100µL of competent cells.
 +
4. sit on ice for 30 minutes
4. sit on ice for 30 minutes
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5. Heat shock at 42ºC for 60 sec in water bath.
5. Heat shock at 42ºC for 60 sec in water bath.
 +
6. Recover on ice for 5 min.
6. Recover on ice for 5 min.
 +
7. Add 300µL of NZYX medium.
7. Add 300µL of NZYX medium.
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8. Incubate with shaking at 37°C for 2hrs. (1hr)
8. Incubate with shaking at 37°C for 2hrs. (1hr)
 +
9. Spin the incubated E.coli suspension for 3min at maximum speed. (Optional)
9. Spin the incubated E.coli suspension for 3min at maximum speed. (Optional)
 +
10. Discard about half of the liquid, and then re-suspend the E.coli solution. (Optional)
10. Discard about half of the liquid, and then re-suspend the E.coli solution. (Optional)
 +
11. Spread the suspension onto selective agar plates.
11. Spread the suspension onto selective agar plates.
 +
12. Incubate at 37°C overnight.
12. Incubate at 37°C overnight.
-
13. Pick a colony into 2ml selective LB (Add 100µL antibiotic to 50mL LB medium). Incubate 37°C in shaker overnight.  
+
 
 +
13. Pick a colony into 2ml selective LB (Add 100µL antibiotic to 50mL LB medium). Incubate 37°C in shaker  
 +
overnight.  
{{:Team:UCL_London/templates/v2/footerFullWidth}}
{{:Team:UCL_London/templates/v2/footerFullWidth}}

Latest revision as of 20:48, 24 October 2010

UCL IGEM 2010

RETURN TO IGEM 2010


NZY + Broth

(per litre) 

  10 g of NZ amine (casein hydrolysate)

  5 g of yeast extract

  5 g of NaCl

Add deionized H2O to a final volume of 1 liter. Adjust to pH 7.5 using NaOH.

Autoclave.

Add the following filer-sterilized supplements prior to use:

12.5 ml of 1 M MgCl2

12.5 ml of 1 M MgSO4

20 ml of 20% (w/v) glucose (or 10 ml of 2 M glucose) Transformation:

Material 

  Plasmids DNA

  Competent E.coli

  NZY Medium

  LB buffer

  Ampicllin, Kanamycin, Tetracycline

  Agar Plates

  Eppendorf


Method

UCL-Plate1.JPG

1. Punch the appropriate well with a clean tip.

2. Soak the spots in 10µL of TE that has been warmed to 50ºC for 20 minutes. At this time take the competent cells from the -80º freezer and start defrosting on ice.

3. Add 4µL of DNA in TE to 100µL of competent cells.

4. sit on ice for 30 minutes

5. Heat shock at 42ºC for 60 sec in water bath.

6. Recover on ice for 5 min.

7. Add 300µL of NZYX medium.

8. Incubate with shaking at 37°C for 2hrs. (1hr)

9. Spin the incubated E.coli suspension for 3min at maximum speed. (Optional)

10. Discard about half of the liquid, and then re-suspend the E.coli solution. (Optional)

11. Spread the suspension onto selective agar plates.

12. Incubate at 37°C overnight.

13. Pick a colony into 2ml selective LB (Add 100µL antibiotic to 50mL LB medium). Incubate 37°C in shaker overnight.


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