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)