Difference between revisions of "Creating Lac- E. coli Mutants"

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1. Prepare solution of bacteria and sterile PBS (depending on date, the O.D might differ) in test tube with green cap
 
1. Prepare solution of bacteria and sterile PBS (depending on date, the O.D might differ) in test tube with green cap
1.5 mL bacteria + 1.5 mL sterile PBS
+
:a. 1.5 mL bacteria + 1.5 mL sterile PBS
Use a blank containing at least 3 mL of PBS, insert into spectrophotometer, and press the arrow up button
+
2. Use a blank containing at least 3 mL of PBS, insert into spectrophotometer, and press the arrow up button
Take out blank and insert test tube into the spectrophotometer
+
3. Take out blank and insert test tube into the spectrophotometer
Check if OD is ~0.4
+
4. Check if OD is ~0.4
If OD is not ~0.4, remove 1 mL of the solution and add 1 mL of either PBS or bacteria depending on whether OD is higher or lower than 0.4. Repeat until OD is ~0.4.
+
5. If OD is not ~0.4, remove 1 mL of the solution and add 1 mL of either PBS or bacteria depending on whether OD is higher or lower than 0.4. Repeat until OD is ~0.4.

Revision as of 04:10, 10 April 2020

Goal

To create Lac negative E. coli mutants for student/faculty use.

Pre-Protocol Questions

1. Do you know how to use the spectrophotometer? The stratalinker?

2. Do you have enough PBS? Do you have enough bacteria?

Protocol

1. Prepare solution of bacteria and sterile PBS (depending on date, the O.D might differ) in test tube with green cap

a. 1.5 mL bacteria + 1.5 mL sterile PBS

2. Use a blank containing at least 3 mL of PBS, insert into spectrophotometer, and press the arrow up button 3. Take out blank and insert test tube into the spectrophotometer 4. Check if OD is ~0.4 5. If OD is not ~0.4, remove 1 mL of the solution and add 1 mL of either PBS or bacteria depending on whether OD is higher or lower than 0.4. Repeat until OD is ~0.4.