Difference between revisions of "Team:Ionis Paris/Protocol 9"

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                                 <br/><p>Centrifuge the bacterial O/N mini-cultures (1 to 5 mL) at 9000 rpm for 3 min at room temperature to pellet cells. Discard the supernatant.<br/></p>
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                                 <br/><p>
−
<p>Resuspend the pellet in 200 μL Plasmid Resuspension Buffer (B1). Vortex or pipet to ensure cells are completely resuspended. There should be no visible clumps.<br/> </p>
+
From the bacterial O/N minicultures, take 500µL that will serve to make bacteria storage at -80°C.
−
<p>Lyse cells by adding 200 μl Plasmid Lysis Buffer (B2). Invert tube immediately and gently 5–6 times until color changes to dark pink and the solution is clear and viscous. Do not vortex! Incubate for 1 min.<br/></p>
+
(See the appropriate protocol, Protocol 11 : Glycerol storage) <br/><br/>
−
<p>Neutralize the lysate by adding 400 μl of Plasmid Neutralization Buffer (B3). Gently invert tube until color is uniformly yellow and a precipitate forms. Do not vortex! Incubate for 2 min.<br/>
+
Centrifuge the remaining bacterial O/N mini-cultures (1 to 5 mL) at 13000 rpm for 1 min at room temperature to pellet cells. Discard the supernatant.<br/></p>
−
Clarify the lysate by spinning for 4 min at 13000 rpm.</p>
+
<p>Resuspend the pellet in 200μL Plasmid Resuspension Buffer (B1). Vortex or pipet to ensure cells are completely resuspended. There should be no visible clumps.<br/> </p>
 +
<p>Lyse cells by adding 200μl Plasmid Lysis Buffer (B2). Invert tube immediately and gently 5–6 times until color changes to dark pink and the solution is clear and viscous. Do not vortex! Incubate for 1 min.<br/></p>
 +
<p>Neutralize the lysate by adding 400μl of Plasmid Neutralization Buffer (B3). Gently invert tube until color is uniformly yellow and a precipitate forms. Do not vortex! Incubate for 2 min.<br/>
 +
Clarify the lysate by spinning for 5 min at 13000 rpm.</p>
 
<p>Carefully transfer supernatant to the spin column and centrifuge for 1 min. Discard flow-through.<br/></p>
 
<p>Carefully transfer supernatant to the spin column and centrifuge for 1 min. Discard flow-through.<br/></p>
−
<p>Re-insert column in the collection tube and add 200 μl of Plasmid Wash Buffer 1. Plasmid Wash Buffer 1 removes RNA, protein and endotoxin. Centrifuge for 1 min. Discard the flow-through. <br/></p>
+
<p>Re-insert column in the collection tube and add 200μl of Plasmid Wash Buffer 1. Plasmid Wash Buffer 1 removes RNA, protein and endotoxin. Centrifuge for 1 min. Discard the flow-through. <br/></p>
−
<p>Add 400 μl of Plasmid Wash Buffer 2 and centrifuge for 1 min. <br/></p>
+
<p>Add 400μl of Plasmid Wash Buffer 2 and centrifuge for 1 min. <br/></p>
 
<p>Transfer column to a clean 1.5 mL microfuge tube. Use care to ensure that the tip of the column has not come into contact with the flow-through. If there is any doubt, re-spin the column for 1 min before inserting it into the clean microfuge tube. <br/></p>
 
<p>Transfer column to a clean 1.5 mL microfuge tube. Use care to ensure that the tip of the column has not come into contact with the flow-through. If there is any doubt, re-spin the column for 1 min before inserting it into the clean microfuge tube. <br/></p>
−
<p>Add ≥ 30 μl DNA Elution Buffer to the center of the matrix. Wait for 1 min, then spin for 1 min to elute DNA.
+
<p>Add 50μl DNA Elution Buffer to the center of the matrix. Wait for 1 min, then spin for 1 min to elute DNA.
 
Store the purified DNA at -20°C</p>
 
Store the purified DNA at -20°C</p>
 
                                      
 
                                      

Revision as of 19:09, 16 October 2016

Protocol 9: Miniprep

Aim: Extract and purify plasmidic DNA from transformed bacteria


From the bacterial O/N minicultures, take 500µL that will serve to make bacteria storage at -80°C. (See the appropriate protocol, Protocol 11 : Glycerol storage)

Centrifuge the remaining bacterial O/N mini-cultures (1 to 5 mL) at 13000 rpm for 1 min at room temperature to pellet cells. Discard the supernatant.

Resuspend the pellet in 200μL Plasmid Resuspension Buffer (B1). Vortex or pipet to ensure cells are completely resuspended. There should be no visible clumps.

Lyse cells by adding 200μl Plasmid Lysis Buffer (B2). Invert tube immediately and gently 5–6 times until color changes to dark pink and the solution is clear and viscous. Do not vortex! Incubate for 1 min.

Neutralize the lysate by adding 400μl of Plasmid Neutralization Buffer (B3). Gently invert tube until color is uniformly yellow and a precipitate forms. Do not vortex! Incubate for 2 min.
Clarify the lysate by spinning for 5 min at 13000 rpm.

Carefully transfer supernatant to the spin column and centrifuge for 1 min. Discard flow-through.

Re-insert column in the collection tube and add 200μl of Plasmid Wash Buffer 1. Plasmid Wash Buffer 1 removes RNA, protein and endotoxin. Centrifuge for 1 min. Discard the flow-through.

Add 400μl of Plasmid Wash Buffer 2 and centrifuge for 1 min.

Transfer column to a clean 1.5 mL microfuge tube. Use care to ensure that the tip of the column has not come into contact with the flow-through. If there is any doubt, re-spin the column for 1 min before inserting it into the clean microfuge tube.

Add 50μl DNA Elution Buffer to the center of the matrix. Wait for 1 min, then spin for 1 min to elute DNA. Store the purified DNA at -20°C