Team:Glendale CC AZ/Protocols/NaCl

From 2013.igem.org

(Difference between revisions)
Line 20: Line 20:
== Procedure: ==
== Procedure: ==
 +
1. Grow E. coli in LB liquid media until stationary phase.
-
1. Grow E. coli in LB liquid media until stationary phase
 
2. To 4 separate spec tubes add:
2. To 4 separate spec tubes add:
     -1st: 5 mL of LB media
     -1st: 5 mL of LB media
     -2nd: 5 mL of LB media supplemented with NaCl to a final concentration of 0.65 M NaCl
     -2nd: 5 mL of LB media supplemented with NaCl to a final concentration of 0.65 M NaCl
     -3rd: 5 mL of LB media supplemented with IPTG
     -3rd: 5 mL of LB media supplemented with IPTG
-
     -4th: 5 mL of LB media supplemented with IPTG and NaCl to a final concentration of 0.65 M NaCl
+
     -4th: 5 mL of LB media supplemented with IPTG and NaCl to a final concentration of 0.65 M NaCl.
 +
 
3. Add 300 uL of bacterial liquid culture to each of the tubes. Use parafilm to seal them  in order to minimize contamination.
3. Add 300 uL of bacterial liquid culture to each of the tubes. Use parafilm to seal them  in order to minimize contamination.
 +
4. Add 5.3 mL of LB media to the last spec tube which will be use to blank the spectrophotometer. Seal with parafilm.
4. Add 5.3 mL of LB media to the last spec tube which will be use to blank the spectrophotometer. Seal with parafilm.
 +
5. Use Kimwipes to clean any prints or smear on the spec tubes. Invert each tube twice before placing in spectrophotometer.
5. Use Kimwipes to clean any prints or smear on the spec tubes. Invert each tube twice before placing in spectrophotometer.
 +
6. Blank the spectrophotometer. Read absorbance of each spec tube.
6. Blank the spectrophotometer. Read absorbance of each spec tube.
 +
7.  Incubate all tubes at 37ºC for 3 hours.
7.  Incubate all tubes at 37ºC for 3 hours.
 +
8. Repeat steps 6-7 after 24 hours.
8. Repeat steps 6-7 after 24 hours.

Revision as of 19:24, 11 August 2013


NaCl Stress Growth Assay

Purpose: To measure bacterial growth with NaCl as DNA damaging agent


Materials: 

       -5 spec tubes
       -Spectrophotometer	
       -Incubator at 37ºC
       -Micropipette
       -Disposable micropipette tips  
       -E. coli in LB media 
       -NaCl
       - 250 mM IPTG


Procedure:

1. Grow E. coli in LB liquid media until stationary phase.

2. To 4 separate spec tubes add: 

    -1st: 5 mL of LB media
    -2nd: 5 mL of LB media supplemented with NaCl to a final concentration of 0.65 M NaCl
    -3rd: 5 mL of LB media supplemented with IPTG
    -4th: 5 mL of LB media supplemented with IPTG and NaCl to a final concentration of 0.65 M NaCl.

3. Add 300 uL of bacterial liquid culture to each of the tubes. Use parafilm to seal them in order to minimize contamination.

4. Add 5.3 mL of LB media to the last spec tube which will be use to blank the spectrophotometer. Seal with parafilm.

5. Use Kimwipes to clean any prints or smear on the spec tubes. Invert each tube twice before placing in spectrophotometer.

6. Blank the spectrophotometer. Read absorbance of each spec tube.

7. Incubate all tubes at 37ºC for 3 hours.

8. Repeat steps 6-7 after 24 hours.

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