Team:Imperial College London/Project/Auxin/Protocols

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(Created page with "<html> <h2>27th of July</h2> The strain of E. coli with the copy of super-folded GFP that was incubated the previous night has shown limited/no growth in the prepared LB broth. T...")
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<b>New protocol:</b><br>
<b>New protocol:</b><br>
50μg/ml is the recommended concentration. We will use 35-40μg/ml of kanamycin to speed up growth.</html>
50μg/ml is the recommended concentration. We will use 35-40μg/ml of kanamycin to speed up growth.</html>
 +
<html>
 +
<h2>28th of July</h2>
 +
Transformation of cells with 6, 7 and 8:<br><br>
 +
-Let competent cell strain 5α thaw for around 10 minutes on ice.<br>
 +
-Add 2-3μl of DNA.<br>
 +
-Leave on ice for 20-25 minutes.<br>
 +
-Heat shock cells at 42°C for 45 seconds.<br>
 +
-Leave on ice for 10 minutes.<br>
 +
-Add 500μl of LB broth.<br>
 +
-Incubate for 1 hour at 37°C.<br>
 +
-Centrifuge for 1 minute.<br>
 +
-Remove 100μl off the top of the eppendorf tube. Pour out the rest making sure that the pellet remains in the eppendorf tube.<br>
 +
-Re-suspend the cells in the 100μl LB broth solution that was removed in the previous step.
 +
-Add 5μl on a chloramphenicol agar plate (concentration of 35μg/ml).<br>
 +
-Add the rest of the sample to a second chloramphenicol agar plate.</html>

Revision as of 13:28, 28 July 2011

27th of July

The strain of E. coli with the copy of super-folded GFP that was incubated the previous night has shown limited/no growth in the prepared LB broth. This could have been caused by:
-No innoculation
-Kanamycin concentration in LB broth was too high. We had used 85μg/ml.

New protocol:
50μg/ml is the recommended concentration. We will use 35-40μg/ml of kanamycin to speed up growth.

28th of July

Transformation of cells with 6, 7 and 8:

-Let competent cell strain 5α thaw for around 10 minutes on ice.
-Add 2-3μl of DNA.
-Leave on ice for 20-25 minutes.
-Heat shock cells at 42°C for 45 seconds.
-Leave on ice for 10 minutes.
-Add 500μl of LB broth.
-Incubate for 1 hour at 37°C.
-Centrifuge for 1 minute.
-Remove 100μl off the top of the eppendorf tube. Pour out the rest making sure that the pellet remains in the eppendorf tube.
-Re-suspend the cells in the 100μl LB broth solution that was removed in the previous step. -Add 5μl on a chloramphenicol agar plate (concentration of 35μg/ml).
-Add the rest of the sample to a second chloramphenicol agar plate.