From 2010.igem.org
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- | '''Day 1:'''
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- | -Inoculate 5 ml [[LB( TY) medium]] with ''E.coli'' from glycerol stock
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- | -Grow overnight at 37 oC
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- | '''Day 2:'''
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- | -Inoculate 20 ml LB medium with 200 μl overnight culture
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- | -Grow at 37 oC until OD600 = 0,3-0,5(+/- 2 hours)
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- | -Spin down 5 minute at 4000 rpm at 4 oC
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- | -Resuspend in 10 ml chilled 0,1 M CaCl2 (from here, keep on ice! )
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- | -Inoculate on ice for 20 minutes
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- | -Spin down as before
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- | -Remove supernatant
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- | -Resuspend in 2 ml 0,1 M CaCl2 /10% glycerol
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- | -Divide 100 μl aliquots
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- | -Store competent cells in - 80 oC
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- | '''Transformation protocol'''
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- | Melt agar in autoclave or microwave. Cool down to 60 oC. One jar ( 100mL ) is enough for 7 plates.
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- | -Add 10 μl of ligation product to 100 μl of cells ( don't for get controls! )
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- | -Incubate 30 min on ice (make agar plates)
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- | -Incubate for 1 min at 42 oC
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- | -Add 400 μl of TY
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- | -Incubate for 30 min at 37 oC (dry agar plates)
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- | -Plate out. Use 250 μl of the transformed cells.
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- | *If you expect the transformation to be very efficient (for instance using undigested plasmid DNA) first make dilution (1:10 or 1:100 ) and plate 250 μl of this
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- | *If you expect the trasformation to be inefficient, concentrate cells by spinning the 500 μl and resuspending the pellet in 250 μl, which you will use for plating.
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Latest revision as of 14:10, 24 August 2010