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From 2012.igem.org

Promoters & Reporters workgroup

Plasmid purification
1) Pellet 3mL bacterial culture by centrifugation at 8000 rpm for 3 min at room temperature
2) Resuspend pelleted bacterial cells in 250 uL Buffer P1
3) Add 250 uL Buffer P2 and mix 4-6 times. Let it for no more than 5 min
4) Add 350 uL Buffer N3 and mix 4-6 times
5) Centrifugation at 13 000 rpm for 10 min
6) Take the supernatant and apply to the QIAprep spin column
7) Centrifugation at 13 000 rpm for 60 sec
8) Discard flow-through
9) Centrifugation at 13 000 rpm for 60 sec
10) Discard flow-through
11) Place column in a clean 1.5 mL microcentrifuge tube
12) Add 50 uL Buffer EB to the column for elution and let stand for 1 min
13) Centrifugation at 13 000 rpm for 1 min

DNA Concentrations
unit= ng/uL
CFP : 240,1 ng/uL
GFP : 209,1 ng/uL
RFP : 142,8 ng/uL
YFP : 196,0 ng/uL
pCS2+ : 362,7 ng/uL

MiniPrep digestion
CFP, GFP, RFP, YFP : EcoRI/SpeI(BcuI)
Supermix: 10uL buffer Red + 1uL BSA + 74uL
Mix for each sample : 21,25uL supermix + 1uL DNA + 0,5uL EcoRI + 0,5uL BcuI
pCS2+ : EcoRI/XbaI
2,5uL buffer Orange + 0,25uL BSA + 18,5uL ddH2O
=> 3h, 37°C