Team:UT-Tokyo/Restriction Enzyme digestion（Xba1/Pst1）—Once for all

From 2010.igem.org

(Difference between revisions)
(New page: __NOTOC__{{UT-Tokyo_CSS3}}{{UT-Tokyo_Head}} <h1>Restriction Enzyme digestion（Xba1/Pst1）—Once for all</h1> <h2>Preparation</h2> *plasmid *10×　buffer (H and M in the freezer) *enz...)
 
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<h3>for extracting gel</h3>
<h3>for extracting gel</h3>
begin with 0.5μl enzyme and add to it if you worry (0.5μl is enough)
begin with 0.5μl enzyme and add to it if you worry (0.5μl is enough)
-
*1. add　?μl　plasmid（30μl for 1000ng）
+
*1. add　?μl　plasmid（30μl for 1000ng）<br />
-
　　　　　3μl　10×　buffer
+
　　　　　3μl　10×　buffer<br />
-
　　　　　0.5μl　enzymeI
+
　　　　　0.5μl　enzymeI<br />
-
　　　　　0.5μl　enzymeII
+
　　　　　0.5μl　enzymeII<br />
-
　　　　　MilliQ　up to 30μl
+
　　　　　MilliQ　up to 30μl<br />
-
*2. incubate at 37 degrees for over 1hour
+
*2. incubate at 37 degrees for over 1hour<br />
(for 3 or 4 hour makes it sure)
(for 3 or 4 hour makes it sure)
<h3>for cut check</h3>
<h3>for cut check</h3>
-
*1. add　1μl　plasmid (regardless of the concentration)
+
*1. add　1μl　plasmid (regardless of the concentration)<br />
-
　　　　　2μl　10×　buffer
+
　　　　　2μl　10×　buffer<br />
-
　　　　　0.5μl　enzymeI
+
　　　　　0.5μl　enzymeI<br />
-
　　　　　0.5μl　enzymeII
+
　　　　　0.5μl　enzymeII<br />
-
　　　　　MilliQ　up to 20μl
+
　　　　　MilliQ　up to 20μl<br />
-
*2. incubate at 37 degrees for over 1hour
+
*2. incubate at 37 degrees for over 1hour<br />
(for 3 or 4 hour makes it sure)
(for 3 or 4 hour makes it sure)
{{UT-Tokyo_Foot}}
{{UT-Tokyo_Foot}}

Latest revision as of 07:37, 23 September 2010

UT-Tokyo

Restriction Enzyme digestion（Xba1/Pst1）—Once for all

Preparation

  • plasmid
  • 10×　buffer (H and M in the freezer)
  • enzymeI,enzymeII（in the freezer in the next room）
  • MilliQ
  • 1.5ml tube

■the correspondence of buffers

EX・・・M　ES・・・H　XP・・・M　SP・・・H

Procedure

■attention

  • use 1.5ml tube
  • enzyme must always be on ice! / Don’t leave at room temperature and heat!

put back in the freezer immediately!

  • thaw frozen buffer fully! / you should spin down and vortex
  • change MilliQ into new one everyday

※add MilliQ first and enzyme last / mix before add enzyme


for extracting gel

begin with 0.5μl enzyme and add to it if you worry (0.5μl is enough)

  • 1. add　?μl　plasmid（30μl for 1000ng）

　　　　　3μl　10×　buffer
　　　　　0.5μl　enzymeI
　　　　　0.5μl　enzymeII
　　　　　MilliQ　up to 30μl

  • 2. incubate at 37 degrees for over 1hour

(for 3 or 4 hour makes it sure)

for cut check

  • 1. add　1μl　plasmid (regardless of the concentration)

　　　　　2μl　10×　buffer
　　　　　0.5μl　enzymeI
　　　　　0.5μl　enzymeII
　　　　　MilliQ　up to 20μl

  • 2. incubate at 37 degrees for over 1hour

(for 3 or 4 hour makes it sure)

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