Team:Glendale CC AZ/Protocols/NaCl

Glendale Community College Arizona

Protocols

Growth Curve Assay

NaCl Growth Curve Assay

Survival Growth Assay

Alkaline Lysis Plasmid Miniprep

Restriction Digest

DNA Isolation

Bioinformatics

Ligation

Transformation

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.