Team:UT-Tokyo/Miniprep

From 2010.igem.org

(Difference between revisions)
(New page: __NOTOC__{{UT-Tokyo_CSS3}}{{UT-Tokyo_Head}} <h1>Miniprep</h1> <h2>Preparation</h2> *kit of Promega (SVMinipreps) *incubative tube *2ml epp tube *1.5ml epp tube *MilliQ <h2>Procedure</h...)
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( you should decant by using yellow tip first / remove culture medium as you can /  
( you should decant by using yellow tip first / remove culture medium as you can /  
throw waste water away in bio hazard!)
throw waste water away in bio hazard!)
-
*4. add 250μl cell resuspension solution (<red>red label</red>)、suspend completely
+
*4. add 250μl cell resuspension solution (<font color="red">red label</font>)、suspend completely
(incomplete suspending decreases yields / you should use epp stand like a washboard)
(incomplete suspending decreases yields / you should use epp stand like a washboard)
-
*5. add 250μl Cell lysis solution（<green>green label</green>）
+
*5. add 250μl Cell lysis solution（<font color="green">green label</font>）
*6. turn the tube upside down four times slowly not to bubble  
*6. turn the tube upside down four times slowly not to bubble  
*7. add 10μl Alkalin　Protease　Sol. （small bottle）
*7. add 10μl Alkalin　Protease　Sol. （small bottle）
*8. turn the tube upside down four times slowly not to bubble
*8. turn the tube upside down four times slowly not to bubble
*9. wait for 5min （Be careful not to exceed 5min! colon bacillus will disintegrate too much!）
*9. wait for 5min （Be careful not to exceed 5min! colon bacillus will disintegrate too much!）
-
*10. add 350μl Neutralization Sol. (<blue>blue label</blue>）
+
*10. add 350μl Neutralization Sol. (<font color="blue">blue label</font>）
*11. turn the tube upside down four times slowly not to bubble
*11. turn the tube upside down four times slowly not to bubble
*12. centrifuge for 10min (15,000rpm)
*12. centrifuge for 10min (15,000rpm)

Revision as of 08:18, 23 September 2010

UT-Tokyo

Miniprep

Preparation

  • kit of Promega (SVMinipreps)
  • incubative tube
  • 2ml epp tube
  • 1.5ml epp tube
  • MilliQ

Procedure

  • 1. pour contents out of the incubative tube into the 1.5ml tube as you can
  • 2. centrifuge for 10min (15,000rpm)
  • 3. throw supernatant fluid away not to damage the precipitation

( you should decant by using yellow tip first / remove culture medium as you can / throw waste water away in bio hazard!)

  • 4. add 250μl cell resuspension solution (red label)、suspend completely

(incomplete suspending decreases yields / you should use epp stand like a washboard)

  • 5. add 250μl Cell lysis solution（green label）
  • 6. turn the tube upside down four times slowly not to bubble
  • 7. add 10μl Alkalin　Protease　Sol. （small bottle）
  • 8. turn the tube upside down four times slowly not to bubble
  • 9. wait for 5min （Be careful not to exceed 5min! colon bacillus will disintegrate too much!）
  • 10. add 350μl Neutralization Sol. (blue label）
  • 11. turn the tube upside down four times slowly not to bubble
  • 12. centrifuge for 10min (15,000rpm)
  • 13. put the supernatant fluid to column (germ’s wreckage is adhering below)
  • 14. centrifuge for 1min (15,000rpm)
  • 15. throw flow through (the liquid in the tube below) away
  • 16. add 750μl Wash Sol. to column and centrifuge for 1min (15,000rpm)
  • 17. throw flow through away, put 250μl Wash Sol. to column and centrifuge for 1min (15,000rpm)
  • 18. change the column into 1.5ml tube and centrifuge for 2min (15,000rpm)
  • 19. change the tube into new one and add 50μl MilliQ

(use Nucleas-Free Water in the kit instead of MilliQ)

  • 20. centrifuge for 1min (15,000rpm) after waiting for 1min
  • 21. take 1 to 1.5μl and determine the concentration by NanoDrop (Don’t dilute)
  • 22. label them


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