Team:UT-Tokyo/Miniprep

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*2. centrifuge for 10min (15,000rpm)
*2. centrifuge for 10min (15,000rpm)
*3. throw supernatant fluid away not to damage the precipitation
*3. throw supernatant fluid away not to damage the precipitation
-
( 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 (<font color="red">red label</font>)、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(<font color="green">green label</font>)
*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  
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*18. change the column into 1.5ml tube and centrifuge for 2min (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
*19. change the tube into new one and add 50μl MilliQ
-
(use Nucleas-Free Water in the kit instead of MilliQ)
+
**(use Nucleas-Free Water in the kit instead of MilliQ)
*20. centrifuge for 1min (15,000rpm) after waiting for 1min
*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)
*21. take 1 to 1.5μl and determine the concentration by NanoDrop (Don’t dilute)

Latest revision as of 08:20, 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