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

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(Created page with "==Goal== To create Lac negative E. coli mutants for student/faculty use. ==Pre-Protocol Questions== 1. Do you know how to properly streak a strain onto a plate using an inocu...")
 
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==Goal==
 
==Goal==
 +
 
To create Lac negative E. coli mutants for student/faculty use.
 
To create Lac negative E. coli mutants for student/faculty use.
 +
 
==Pre-Protocol Questions==
 
==Pre-Protocol Questions==
  
1. Do you know how to properly streak a strain onto a plate using an inoculating loop?
+
1. Do you know how to use the spectrophotometer? The stratalinker?
  
2. Do you know how to pick a single colony from a plate?
+
2. Do you have enough PBS? Do you have enough bacteria?
  
3. Do you have liquid LB media? sterile Erlenmeyer flasks? milliQ water? virkon solution? any other materials you will need for this protocol?
+
==Protocol==
  
4. Do you know how to use the Plasmid Miniprep Kit? the NanoDrop Machine?
+
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
==Protocol==
+
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
 +
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.

Revision as of 04:07, 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 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 Take out blank and insert test tube into the spectrophotometer 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.