Team:USTC CHINA/Notebook/Protocols/Extracting plasmids from gram-positive bacterium

From 2013.igem.org

(Difference between revisions)
(Created page with "{{USTC-China/hidden}} <html> <head> <link rel="stylesheet" type="text/css" href="https://2013.igem.org/Team:USTC_CHINA/main.css?action=raw&ctype=text/css" /> </head> <body backgro...")
Line 53: Line 53:
<div class="content" align="center">
<div class="content" align="center">
<div class="conbar1">
<div class="conbar1">
-
<div id="breadcrumb"><a href="https://2013.igem.org/Team:USTC_CHINA">Home</a> &gt; <a href="https://2013.igem.org/Team:USTC_CHINA/Notebook">notebook</a> &gt; <a href="https://2013.igem.org/Team:USTC_CHINA/Notebook/Protocols">protocols</a></div></div>
+
<div id="breadcrumb"><a href="https://2013.igem.org/Team:USTC_CHINA">Home</a> &gt; <a href="https://2013.igem.org/Team:USTC_CHINA/Notebook">notebook</a> &gt; <a href="https://2013.igem.org/Team:USTC_CHINA/Notebook/Protocols">protocols</a>&gt; <a href="https://2013.igem.org/Team:USTC_CHINA/Notebook/Protocols/Extracting plasmids from gram-positive ">protocols Extracting plasmids from gram-positive </a></div></div>
<div class="conbar2">
<div class="conbar2">
<div class="leftbar" align="left">
<div class="leftbar" align="left">
<div class="bassic-bar">
<div class="bassic-bar">
-
<h1>Gel Extraction</h1>
+
<h1>Extracting plasmids from gram-positive </h1>
-
<p>Performed with AxyPrep 96-well DNA Gel Extraction Kit (type: AP-96-GX)
+
<p>1.Centrifuge 2-3ml bacteria liquid with 8000rpm and remove the supernatant; resuspense the liquid with lysyme(10mg/ml)~200ul and then leave it at temperature 37℃ for 0.5-1 hour.</br>
-
Protocol
+
2.Centrifuge the liquid with 8000rpm and remove the supernatant; resuspense it with 250uL buffer P1.</br>
-
1. Excise gel slice containing DNA fragment of interest.
+
3.Add 250uL buffer P2 (Alkaline lysis buffer), make it clear (reverse the tube evenly for several times) and leave it at room temperature for 2-4 minutes.</br>
-
2. Add 3×sample volume of Buffer DE-A.
+
4.Add 350uL buffer P3 and reverse the tube evenly until it layers.</br>
-
Incubate at 75° C for 15-20 min or until gel melts completely.
+
5.Centrifuge the tube for 10 minutes and extract plasmid extraction column from the upper water phase.</br>
-
Add 0.5 × Buffer DE-A volume of Buffer DE-B.
+
6.The following steps are identical with those of Escherichia coli.</br>
-
<img src="https://static.igem.org/mediawiki/2013/4/4d/2013igemustc-china_Protocol1.png" width="580" height="200" />  
+
 
-
3. Binding sample DNA(Centrifuge at 5,000 - 6,000×g for 5 min)
+
-
+
-
4. Add 800 μl of Buffer W2 (5,000 - 6,000×g for 1 min)
+
-
Repeat wash with Buffer W2
+
-
Centrifuge empty plate for 5 min at 5,000 - 6,000×g to remove residue W2.
+
-
+
-
5. Elute with 50 μl of Eluent or Deionized Water.(5,000 - 6,000×g, 5min)
+
-
+
</p></div>
</p></div>

Revision as of 17:37, 25 September 2013

Extracting plasmids from gram-positive

1.Centrifuge 2-3ml bacteria liquid with 8000rpm and remove the supernatant; resuspense the liquid with lysyme(10mg/ml)~200ul and then leave it at temperature 37℃ for 0.5-1 hour.
2.Centrifuge the liquid with 8000rpm and remove the supernatant; resuspense it with 250uL buffer P1.
3.Add 250uL buffer P2 (Alkaline lysis buffer), make it clear (reverse the tube evenly for several times) and leave it at room temperature for 2-4 minutes.
4.Add 350uL buffer P3 and reverse the tube evenly until it layers.
5.Centrifuge the tube for 10 minutes and extract plasmid extraction column from the upper water phase.
6.The following steps are identical with those of Escherichia coli.

notebook

Retrieved from "http://2013.igem.org/Team:USTC_CHINA/Notebook/Protocols/Extracting_plasmids_from_gram-positive_bacterium"