iGEM UT-Tokyo

Preparation

iGEM parts / ligation products

SOC or LB (No antibiotic)　500μL

TE　15μL

plates

ice box

heat block(42°C)

competent cells

→ always on ice! Melt on ice! Mix DNA as soon as cells melt!

Protocol

to thaw out igem parts

1. With a pipette tip, punch a hole in the foil
2. Add 15uL of TE (MilliQ),and pipetting
3. Pipette 1uL of the resuspended DNA Transformation into your desired competent cells
4. Hold on ice for 30 mins
5. Heat shock at 42°C for 45 seconds (and on ice after it)
6. Add 300uL of LBborth in each epp
7. Wait for 10 mins
8. Hold at 37℃ for 30 mins

   (this step can be skipped with ampicillin selection)

9. Plate out
10. Incubate at 37°C

Preparation

kit of Promega (SVMinipreps)

incubative 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)

   (you can centrifuge incubative tube directly when it can endure up to 6,000g )

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