Team:Oxford/protocols/MiniPrep: Plasmid Extraction

From 2014.igem.org

(Difference between revisions)
 
(11 intermediate revisions not shown)
Line 3: Line 3:
<div class="abcdefg">
<div class="abcdefg">
<div class="container cf row">
<div class="container cf row">
-
<h1>MiniPrep plasmid extraction protocol</h1>
+
<h1>QIAprep Plasmid DNA Purification</h1>
 +
<p>&#8617; Back to other <html><a href="https://2014.igem.org/Team:Oxford/protocols">protocols</a></html>.</p>
 +
<!--this will number your steps 1,2,3,etc-->
<ol  style="list-style-type:decimal;">  
<ol  style="list-style-type:decimal;">  
-
<li>Thaw the DH5-alpha cells (from -80ºC freezer), plasmid sample (from -20ºC freezer), and the antibiotic stock (from -20ºC freezer) ON ICE.</li>
+
<li>After growing cell cultures overnight in LB, resuspend pelleted cells in 250 μl Buffer P1 and transfer it into a microcentrifuge tube. Make sure that RNase A has been added to Buffer P1 before.<BR><BR>
-
<li>Split the thawed cells into 2x 100µl aliquots. </li>
+
<li>Add 250 μl of Buffer P2 and mix gently by inverting the tube 6 times. The cell suspension should turn blue. Try to carry on with the next step after less than 5 minutes to make sure that the lysis reaction doesn't proceed for too long.<BR><BR>
-
<li>To each aliquot add 1µl of plasmid DNA.</li>
+
<li>Add 350 μl of Buffer N3 and mix the suspension immediately by inverting 6 times.<BR><BR>
-
<li>Incubate ON ICE for ~30 minutes. Incubation can be longer than this but certainly NO shorter. </li>
+
<li>Centrifuge for 10 minutes at 13000 rpm (or 17900 x g). Apply the supernatants to a spin colums<BR><BR>
-
<li>During this incubation time prepare the antibiotic agar plates: </li>
+
<li>Centrifuge again for 30-60 seconds and discard the flow-through.<BR><BR>
 +
<li>Wash the spin column by adding 500 μl of the PB buffer and centrifuge for 30-60s.<BR><BR>
 +
<li>Discard the flow-through and wash the spin column again by adding 750 μl of PE buffer. Centrifuge for 1 minute.<BR><BR>
 +
<li>Discard the flow-through and centrifuge again for 1 minute. Now place the column in a 1.5ml microcentrifuge tube.<BR><BR>
 +
<li>Add 50 μl of the EB buffer to the centre of the spin column. Let stand for 2 minutes and centrifuge for 1 minute.<BR><BR>
-
<ol  style="list-style-type:decimal; margin-left:2cm;">
 
-
<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.
 
-
<li>Cool the agar bottle in 67ºC water bath. During this time switch on the the laminar flow hood to create a sterile environment.
 
-
<li>Once cool take the agar and petri dishes to the laminar flow hood.
 
-
<li>Add the appropriate volume of antibiotic to the agar bottle before pouring.
 
-
<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.
 
-
<li>Leave the agar plates to set (~15-30 minutes)
 
-
</ol>
 
-
<li>Heat shock the bacteria by placing them in the 42ºC water bath for NORE MORE THAN 45 SECONDS.
 
-
<li>Immediately place the bacteria on ICE for 1 minute.
 
-
<li>Add 800µl LB and incubate at 37ºC for 1 hour.
 
-
<li>Plate the bacteria under sterile conditions (bunsen burner ON):
 
-
<ol  style="list-style-type:decimal; margin-left:2cm;">
 
-
<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>
 
-
 
-
<li>Incubate at 37ºC overnight. </li>
 
-
 
-
 
-
 
-
</div>
 
{{:Team:Oxford/templates/footer}}
{{:Team:Oxford/templates/footer}}
-
</div>
 

Latest revision as of 15:11, 27 July 2014

QIAprep Plasmid DNA Purification

↩ Back to other protocols.

  1. After growing cell cultures overnight in LB, resuspend pelleted cells in 250 μl Buffer P1 and transfer it into a microcentrifuge tube. Make sure that RNase A has been added to Buffer P1 before.

  2. Add 250 μl of Buffer P2 and mix gently by inverting the tube 6 times. The cell suspension should turn blue. Try to carry on with the next step after less than 5 minutes to make sure that the lysis reaction doesn't proceed for too long.

  3. Add 350 μl of Buffer N3 and mix the suspension immediately by inverting 6 times.

  4. Centrifuge for 10 minutes at 13000 rpm (or 17900 x g). Apply the supernatants to a spin colums

  5. Centrifuge again for 30-60 seconds and discard the flow-through.

  6. Wash the spin column by adding 500 μl of the PB buffer and centrifuge for 30-60s.

  7. Discard the flow-through and wash the spin column again by adding 750 μl of PE buffer. Centrifuge for 1 minute.

  8. Discard the flow-through and centrifuge again for 1 minute. Now place the column in a 1.5ml microcentrifuge tube.

  9. Add 50 μl of the EB buffer to the centre of the spin column. Let stand for 2 minutes and centrifuge for 1 minute.

Retrieved from "http://2014.igem.org/Team:Oxford/protocols/MiniPrep:_Plasmid_Extraction"