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Plasmid Mini Prep
From 2010.igem.org
(New page: Mini Prep 1.Isolate single colony from a streaked plate and inoculate a culture. a.Incubate for ~ 12-16 hrs 2.Pellet bacterial cells by centri...) |
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1.Isolate single colony from a streaked plate and inoculate a culture. | 1.Isolate single colony from a streaked plate and inoculate a culture. | ||
a.Incubate for ~ 12-16 hrs | a.Incubate for ~ 12-16 hrs | ||
Latest revision as of 14:36, 20 July 2010
1.Isolate single colony from a streaked plate and inoculate a culture.
a.Incubate for ~ 12-16 hrs
2.Pellet bacterial cells by centrifugation at 10,000 x g for 1 min
3.Resuspend bacterial pellet by adding 250 ul of Solution I/RNase A solution
4.Add 250 ul of Solution II and mix by inverting tube several times
5.Add 350 ul of Solution III and mix immediately by inverting several times until white precipitate forms.
6.Centrifuge at > 13,000 x g for 10 minutes at room temperature.
7.Prepare HiBind DNA miniprep Column (I) by adding 100 ul of Equilibration Buffer placed in a 2 mL collection tube.
a.Centrifuge for 60 seconds.
b.Discard flow-through liquid
8.Add the cleared supernatant by carefully aspirating it into Column (I) assembled in the 2 mL collection tube.
a.Centrifuge for 1 min
9.Discard flow-through liquid and add 500 ul of Buffer HB to wash the HiBind DNA Miniprep Column (I)
a.Centrifuge for 1 min
b.Discard flow through
10.Discard flow-through liquid and add 700 ul of DNA Wash Buffer diluted with absolute ethanol to wash the HiBind DNA Miniprep Column (I).
a.Centrifuge for 1 min
b.Discard flow through
11.Centrifuge the empty column for 2 min to dry the column matrix. DO NOT skip this step.
12.Place column into clean 1.5 mL microcentrifuge tube. Add 30 to 100 ul of Elution buffer directly onto column matrix.
a.Centrifuge for 1 min to elute DNA
