Team:Oxford/protocols/MiniPrep: Plasmid Extraction

From 2014.igem.org

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<h1>MiniPrep plasmid extraction protocol</h1>
<h1>MiniPrep plasmid extraction protocol</h1>
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<p> 1) Thaw the DH5-alpha cells (from -80ºC freezer), plasmid sample (from -20ºC freezer), and the antibiotic stock (from -20ºC freezer) ON ICE. </p>
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<OL>
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<p> 2) Split the thawed cells into 2x 100µl aliquots. </p>
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<LI>Thaw the DH5-alpha cells (from -80ºC freezer), plasmid sample (from -20ºC freezer), and the antibiotic stock (from -20ºC freezer) ON ICE.
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<p> 3) To each aliquot add 1µl of plasmid DNA.</p>
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<LI>Split the thawed cells into 2x 100µl aliquots.  
-
<p> 4) Incubate ON ICE for ~30 minutes. Incubation can be longer than this but certainly NO shorter. </p>
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<LI>To each aliquot add 1µl of plasmid DNA.
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<p> 5) During this incubation time prepare the antibiotic agar plates: </p>
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<LI>Incubate ON ICE for ~30 minutes. Incubation can be longer than this but certainly NO shorter.  
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<p> 1) Melt the agar jar for 15 minutes with the microwave on ‘defrost’. Check on the bottle every 5 or so minutes to ensure it is not overflowing. </p>
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<LI>During this incubation time prepare the antibiotic agar plates: </OL>
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<p> 2) Cool the agar bottle in 67ºC water bath. During this time switch on the the laminar flow hood to create a sterile environment. </p>
+
 
-
<p> 3) Once cool take the agar and petri dishes to the laminar flow hood. </p>
+
<OL>
-
<p> 4) Add the appropriate volume of antibiotic to the agar bottle before pouring. </p>
+
<LI>Melt the agar jar for 15 minutes with the microwave on ‘defrost’. Check on the bottle every 5 or so minutes to ensure it is not overflowing.
-
<p> 5) Pour the agar evenly to no higher than the raised line. NOTE: place the lid resting on the edge of the dish so as to catch any drops that fall from the bottle. </p>
+
<LI>Cool the agar bottle in 67ºC water bath. During this time switch on the the laminar flow hood to create a sterile environment.
-
<p> 6) Leave the agar plates to set (~15-30 minutes)</p>
+
<LI>Once cool take the agar and petri dishes to the laminar flow hood.
-
<p> 6) Heat shock the bacteria by placing them in the 42ºC water bath for NORE MORE THAN 45 SECONDS. </p>
+
<LI>Add the appropriate volume of antibiotic to the agar bottle before pouring.
-
<p> 7) Immediately place the bacteria on ICE for 1 minute. </p>
+
<LI>Pour the agar evenly to no higher than the raised line. NOTE: place the lid resting on the edge of the dish so as to catch any drops that fall from the bottle.
-
<p> 8) Add 800µl LB and incubate at 37ºC for 1 hour. </p>
+
<LI>Leave the agar plates to set (~15-30 minutes)</OL>
-
<p> 9) Plate the bacteria under sterile conditions (bunsen burner ON):</p>
+
 
-
<p> 1) Place a glass spreader in ethanol. </p>
+
<OL>
-
<p> 2) Flame the spreader to burn off the ethanol and let it cool. </p>
+
<LI>Heat shock the bacteria by placing them in the 42ºC water bath for NORE MORE THAN 45 SECONDS.  
-
<p> 3) Plate #1: spread 100µl of cell culture onto the plate</p>
+
<LI>Immediately place the bacteria on ICE for 1 minute.  
-
<p> 4) Plate #2: spin down the remaining cells from step 3. Discard the supernatant and resuspend the pellet in 100µl of fresh culture. Spread this onto a second plate as in previous step</p>
+
<LI>Add 800µl LB and incubate at 37ºC for 1 hour.  
-
<p> 10) Incubate at 37ºC overnight. </p>
+
<LI>Plate the bacteria under sterile conditions (bunsen burner ON): </OL>
 +
 
 +
<OL>
 +
<LI>Place a glass spreader in ethanol.  
 +
<LI>Flame the spreader to burn off the ethanol and let it cool.  
 +
<LI>Plate #1: spread 100µl of cell culture onto the plate
 +
<LI>Plate #2: spin down the remaining cells from step 3. Discard the supernatant and resuspend the pellet in 100µl of fresh culture. Spread this onto a second plate as in previous step </OL>
 +
<OL><LI>Incubate at 37ºC overnight. </OL>

Revision as of 10:58, 17 July 2014

MiniPrep plasmid extraction protocol

  1. Thaw the DH5-alpha cells (from -80ºC freezer), plasmid sample (from -20ºC freezer), and the antibiotic stock (from -20ºC freezer) ON ICE.
  2. Split the thawed cells into 2x 100µl aliquots.
  3. To each aliquot add 1µl of plasmid DNA.
  4. Incubate ON ICE for ~30 minutes. Incubation can be longer than this but certainly NO shorter.
  5. During this incubation time prepare the antibiotic agar plates:
  1. Melt the agar jar for 15 minutes with the microwave on ‘defrost’. Check on the bottle every 5 or so minutes to ensure it is not overflowing.
  2. Cool the agar bottle in 67ºC water bath. During this time switch on the the laminar flow hood to create a sterile environment.
  3. Once cool take the agar and petri dishes to the laminar flow hood.
  4. Add the appropriate volume of antibiotic to the agar bottle before pouring.
  5. Pour the agar evenly to no higher than the raised line. NOTE: place the lid resting on the edge of the dish so as to catch any drops that fall from the bottle.
  6. Leave the agar plates to set (~15-30 minutes)
  1. Heat shock the bacteria by placing them in the 42ºC water bath for NORE MORE THAN 45 SECONDS.
  2. Immediately place the bacteria on ICE for 1 minute.
  3. Add 800µl LB and incubate at 37ºC for 1 hour.
  4. Plate the bacteria under sterile conditions (bunsen burner ON):
  1. Place a glass spreader in ethanol.
  2. Flame the spreader to burn off the ethanol and let it cool.
  3. Plate #1: spread 100µl of cell culture onto the plate
  4. Plate #2: spin down the remaining cells from step 3. Discard the supernatant and resuspend the pellet in 100µl of fresh culture. Spread this onto a second plate as in previous step
  1. Incubate at 37ºC overnight.


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