Team:TU Darmstadt/protocols/Cell counting/plating

From 2013.igem.org

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<B> Procedure<br></B>
<B> Procedure<br></B>
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1. Fill each tube in the dilution with 90 &mu;l of LB<br>
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<ul style="margin-left:50px; margin-right:50px; text-align:justify; ">
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2. Add 10 &mu;l of the sample to the first tube and mix<br>
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3. From the first tube, remove 10 &mu;l and mix into second tube<br>
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<li>Fill each tube in the dilution with 90 &mu;l of LB</li>
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4. Repeat for the number of dilutions you wish to do (8 should be more than enough)<sup>[2]</sup><br>
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<li>Add 10 &mu;l of the sample to the first tube and mix</li>
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5. Take 10 &mu;l from each dilution and spot it on to the agar plate<br>
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<li>From the first tube, remove 10 &mu;l and mix into second tube</li>
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6. Allow droplet to dry and incubate<br>
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<li>Repeat for the number of dilutions you wish to do (8 should be more than enough)<sup>[2]</sup></li>
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<li>Take 10 &mu;l from each dilution and spot it on to the agar plate</li>
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<li>Allow droplet to dry and incubate</li>
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The first dilutions will contain a thick lawn of cells and the last dilutions will contain no cells. There should be one drop which contains countable single colonies. From this, you can calculate the number of cells in the original sample. For example, if there 4 colonies on dilution 5, there are 4E4 cells/&mu;l.<br>
The first dilutions will contain a thick lawn of cells and the last dilutions will contain no cells. There should be one drop which contains countable single colonies. From this, you can calculate the number of cells in the original sample. For example, if there 4 colonies on dilution 5, there are 4E4 cells/&mu;l.<br>

Revision as of 20:42, 3 October 2013





Cell counting/plating

Materials
 LB agar plate
LB media
tubes

Procedure
 1. Fill each tube in the dilution with 90 μl of LB
2. Add 10 μl of the sample to the first tube and mix
3. From the first tube, remove 10 μl and mix into second tube
4. Repeat for the number of dilutions you wish to do (8 should be more than enough)[2]
5. Take 10 μl from each dilution and spot it on to the agar plate
6. Allow droplet to dry and incubate

The first dilutions will contain a thick lawn of cells and the last dilutions will contain no cells. There should be one drop which contains countable single colonies. From this, you can calculate the number of cells in the original sample. For example, if there 4 colonies on dilution 5, there are 4E4 cells/μl.

References
http://openwetware.org/wiki/Cell_counting/plating

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