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