Difference between revisions of "Team:ICT-Mumbai/Protocols"

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1. Overnight E. coli culture<br>
 
1. Overnight E. coli culture<br>
 
2. 10 ml LB medium in culture tubes<br>
 
2. 10 ml LB medium in culture tubes<br>
−
3. 0.1 M CaCl2
+
3. 0.1 M CaCl2<br>
−
4. 0.1 M CaCl2 containing 25% glycerol
+
4. 0.1 M CaCl2 containing 25% glycerol<br><br>
  
−
<i>Equipment</i>
+
<i>Equipment</i><br>
−
1. Spectrophotometer or plate reader
+
1. Spectrophotometer or plate reader<br>
−
2. Centrifuge
+
2. Centrifuge<br><br>
  
−
<i>Procedure</i>
+
<i>Procedure</i><br>
−
1. Subculture 100 µl overnight grown culture of E. coli in 10 ml LB medium. Shake at 200 rpm at 37°C.
+
1. Subculture 100 µl overnight grown culture of E. coli in 10 ml LB medium. Shake at 200 rpm at 37°C.<br>
−
2. After ~2 hours, using either a spectrophotometer or a plate reader, measure absorbance of the culture at 600 nm. At OD600 = 0.2, harvest the cells by centrifugation.
+
2. After ~2 hours, using either a spectrophotometer or a plate reader, measure absorbance of the culture at 600 nm. At OD600 = 0.2, harvest the cells by centrifugation.<br>
−
3. Wash cells with ice cold solution of 0.1 M CaCl2 two to three times.
+
3. Wash cells with ice cold solution of 0.1 M CaCl2 two to three times.<br>
 
4. Resuspend cells in 100 μl of 0.1 M CaCl2 containing 25% glycerol and store at –80°C till further use.
 
4. Resuspend cells in 100 μl of 0.1 M CaCl2 containing 25% glycerol and store at –80°C till further use.
 
</p>
 
</p>

Revision as of 15:18, 29 October 2017

ICT-Mumbai 2017

Materials
1. Overnight E. coli culture
2. 10 ml LB medium in culture tubes
3. 0.1 M CaCl2
4. 0.1 M CaCl2 containing 25% glycerol

Equipment
1. Spectrophotometer or plate reader
2. Centrifuge

Procedure
1. Subculture 100 µl overnight grown culture of E. coli in 10 ml LB medium. Shake at 200 rpm at 37°C.
2. After ~2 hours, using either a spectrophotometer or a plate reader, measure absorbance of the culture at 600 nm. At OD600 = 0.2, harvest the cells by centrifugation.
3. Wash cells with ice cold solution of 0.1 M CaCl2 two to three times.
4. Resuspend cells in 100 μl of 0.1 M CaCl2 containing 25% glycerol and store at –80°C till further use.

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