Team:Paris Saclay/mini maxi

From 2013.igem.org

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= '''Mini et maxi preparation''' =
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= '''Plasmidic DNA Mini and Maxi preparation''' =
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1. Centrifuge during 1 minute the cellular culture in tubes of 1.5 or 2.0 mL at maximal speed  
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1. Centrifuge during 1 minute the cell culture in tubes of 1.5 or 2.0 mL at maximal speed in a benchtop centrifuge
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2. Add 100µL of solution n°1 (50 mM glucose, 10 mM EDTA and 25 mM Tris pH 8.0) and hang up the residue (culot)
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2. Add 100µL of solution n°1 (50 mM glucose, 10 mM EDTA and 25 mM Tris pH 8.0) and resuspend the cells
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3. Add 200µL of solution 2, buffer solution of lyse (0.2 mM NaOH et 1% SDS), mix the final solution until it is translucent
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3. Add 200µL of solution 2, the lysis solution(0.2 mM NaOH et 1% SDS), mix the final solution until it is translucent
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4. Add 150µL of solution 3, precipitation with potassium acetate 3.3M, incubate in ice during 10min
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4. Add 150µL of solution 3 (potassium acetate 3.3M)to precipitate cell debris, genomic DNA and proteins, and incubate in ice during 10min
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5. Centrifugation at maximal speed during 10 min
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5. Centrifuge at maximal speed during 10 min in a benchtop centrifuge
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6. Take off the supernatant liquid and transfer it in a new tube
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6. Take off the liquid supernatant and transfer it in a new 1.5 ml tube
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7. Add 500µL of phenol chloroforme
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7. Add 500µL of phenol/chloroform/isoamyl alcohol 25/24/1 and mix vigorously
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8. Centrifuge at maximal speed during 8 min, take off the aqueous phase
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8. Centrifuge at maximal speed during 8 min, take off the aqueous phase and transfer it in a new 1.5 ml tube
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9. Precipitation with ethanol  
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9. Precipitate the plasmidic DNA with ethanol (see protocol)
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For Maxi Preparation, the protocol was scaled up for cultures of 50 or 100 ml.
{{Team:Paris_Saclay/incl_fin}}
{{Team:Paris_Saclay/incl_fin}}

Latest revision as of 17:07, 27 September 2013

Navigation Home Team Project Labwork Notebook Human practices

Plasmidic DNA Mini and Maxi preparation

1. Centrifuge during 1 minute the cell culture in tubes of 1.5 or 2.0 mL at maximal speed in a benchtop centrifuge

2. Add 100µL of solution n°1 (50 mM glucose, 10 mM EDTA and 25 mM Tris pH 8.0) and resuspend the cells

3. Add 200µL of solution 2, the lysis solution(0.2 mM NaOH et 1% SDS), mix the final solution until it is translucent

4. Add 150µL of solution 3 (potassium acetate 3.3M)to precipitate cell debris, genomic DNA and proteins, and incubate in ice during 10min

5. Centrifuge at maximal speed during 10 min in a benchtop centrifuge

6. Take off the liquid supernatant and transfer it in a new 1.5 ml tube

7. Add 500µL of phenol/chloroform/isoamyl alcohol 25/24/1 and mix vigorously

8. Centrifuge at maximal speed during 8 min, take off the aqueous phase and transfer it in a new 1.5 ml tube

9. Precipitate the plasmidic DNA with ethanol (see protocol)


For Maxi Preparation, the protocol was scaled up for cultures of 50 or 100 ml.


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