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Team:Evry-Genopole/Notebook/June/28
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<center><h1>Promoters & Reporters workgroup</h1></center> | <center><h1>Promoters & Reporters workgroup</h1></center> | ||
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| + | <FONT SIZE=3>'''Plasmid purification'''</FONT> <br/> | ||
| + | 1) Pellet 3mL bacterial culture by centrifugation at 8000 rpm for 3 min at room temperature<br/> | ||
| + | 2) Resuspend pelleted bacterial cells in 250 uL Buffer P1<br/> | ||
| + | 3) Add 250 uL Buffer P2 and mix 4-6 times. Let it for no more than 5 min<br/> | ||
| + | 4) Add 350 uL Buffer N3 and mix 4-6 times<br/> | ||
| + | 5) Centrifugation at 13 000 rpm for 10 min<br/> | ||
| + | 6) Take the supernatant and apply to the QIAprep spin column<br/> | ||
| + | 7) Centrifugation at 13 000 rpm for 60 sec <br/> | ||
| + | 8) Discard flow-through<br/> | ||
| + | 9) Centrifugation at 13 000 rpm for 60 sec<br/> | ||
| + | 10) Discard flow-through<br/> | ||
| + | 11) Place column in a clean 1.5 mL microcentrifuge tube<br/> | ||
| + | 12) Add 50 uL Buffer EB to the column for elution and let stand for 1 min<br/> | ||
| + | 13) Centrifugation at 13 000 rpm for 1 min<br/> | ||
| + | <br/> | ||
| + | <FONT SIZE=3>'''DNA Concentrations'''</FONT> <br/> | ||
| + | unit= ng/uL<br/> | ||
| + | CFP : 240,1 ng/uL<br/> | ||
| + | GFP : 209,1 ng/uL<br/> | ||
| + | RFP : 142,8 ng/uL<br/> | ||
| + | YFP : 196,0 ng/uL<br/> | ||
| + | pCS2+ : 362,7 ng/uL<br/> | ||
| + | <br/> | ||
<FONT SIZE=3>'''MiniPrep digestion'''</FONT> <br/> | <FONT SIZE=3>'''MiniPrep digestion'''</FONT> <br/> | ||
'''CFP, GFP, RFP, YFP : EcoRI/SpeI(BcuI)'''<br/> | '''CFP, GFP, RFP, YFP : EcoRI/SpeI(BcuI)'''<br/> | ||
Revision as of 16:55, 29 June 2012
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: 10microL buffer Red + 1microL BSA + 74microL
Mix for each sample : 21,25microL supermix + 1microL DNA + 0,5microL EcoRI + 0,5microL BcuI
pCS2+ : EcoRI/XbaI
2,5microL buffer Orange + 0,25microL BSA + 18,5microL ddH2O
=> 3h, 37°C
