Team:USTC CHINA/Notebook/Protocols/Plasmid mini-prep

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 58: Line 58:
<div class="bassic-bar">
<div class="bassic-bar">
<h1>Gel Extraction</h1>
<h1>Gel Extraction</h1>
-
<p>Performed with AxyPrep 96-well DNA Gel Extraction Kit (type: AP-96-GX)
+
<p>Performed with Shanghai Sangon Plasmid miniprep kit.
-
Protocol
+
1. Preparation
-
1. Excise gel slice containing DNA fragment of interest.
+
1) Make sure the RNase A has been added into Buffer P1;
-
2. Add 3×sample volume of Buffer DE-A.
+
2) Make sure the ethanol has been added into Wash Solution
-
Incubate at 75° C for 15-20 min or until gel melts completely.
+
3) Make sure there is no deposit in Buffer P2 and P3
-
Add 0.5 × Buffer DE-A volume of Buffer DE-B.
+
2. Collect the deposit of bacteria from 1.5ml~5ml bacteria liquid (centrifuge at 8,000×g for 2 min)
-
<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)
+
3. Add 250μl Buffer P1 into the deposit and suspend it thoroughly
-
+
4. Add 250μl Buffer P2, mix gently and keep standing in room temperature for 2~4min
-
4. Add 800 μl of Buffer W2 (5,000 - 6,000×g for 1 min)
+
5. Add 350μl Buffer P3 and mix gently for 5~10 times
-
Repeat wash with Buffer W2
+
6. Precipitate the bacteria fragments (centrifuge at 12,000×g for 5~10 min) and extract the supernatant (centrifuge at 8,000×g for 30s)
-
Centrifuge empty plate for 5 min at 5,000 - 6,000×g to remove residue W2.
+
7. Add 500 μl Buffer DW1, centrifuge at 9,000×g for 30s
-
+
8. Add 500 μl Wash Solution, centrifuge at 9,000×g for 30s. Repeat this process for one more time. Centrifuge the empty bottle at 9,000×g for 60s.
-
5. Elute with 50 μl of Eluent or Deionized Water.(5,000 - 6,000×g, 5min)
+
9. Add 50~100μl Elution Buffer and collect the plasmid.
-
+
 
</p></div>
</p></div>

Revision as of 13:41, 25 September 2013

Gel Extraction

Performed with Shanghai Sangon Plasmid miniprep kit. 1. Preparation 1) Make sure the RNase A has been added into Buffer P1; 2) Make sure the ethanol has been added into Wash Solution 3) Make sure there is no deposit in Buffer P2 and P3 2. Collect the deposit of bacteria from 1.5ml~5ml bacteria liquid (centrifuge at 8,000×g for 2 min) 3. Add 250μl Buffer P1 into the deposit and suspend it thoroughly 4. Add 250μl Buffer P2, mix gently and keep standing in room temperature for 2~4min 5. Add 350μl Buffer P3 and mix gently for 5~10 times 6. Precipitate the bacteria fragments (centrifuge at 12,000×g for 5~10 min) and extract the supernatant (centrifuge at 8,000×g for 30s) 7. Add 500 μl Buffer DW1, centrifuge at 9,000×g for 30s 8. Add 500 μl Wash Solution, centrifuge at 9,000×g for 30s. Repeat this process for one more time. Centrifuge the empty bottle at 9,000×g for 60s. 9. Add 50~100μl Elution Buffer and collect the plasmid.

notebook

Retrieved from "http://2013.igem.org/Team:USTC_CHINA/Notebook/Protocols/Plasmid_mini-prep"