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Team:KIT-Kyoto/nf08123
From 2013.igem.org
Protocol
Miniprep
Solution Ⅰ (50 mM Tris-HCl and 10 mM EDTA, and 50 µg/mL RNase A, pH 8.0 (25˚C))
Solution Ⅱ (0.2 M NaOH and 1 % SDS)
Solution Ⅲ (4 M guanidine hydrochloride and 0.5 M potassium acetate, pH 4.2)
Solution Ⅳ (5 M guanidine hydrochloride, 20 mM Tris-HCl, and 38% ethanol, pH 6.6 (25˚C))
Solution Ⅴ (2 mM Tris-HCl and 80% ethanol, pH 7.5 (25˚C))
Pick up a single colony from plate and cultivate it overnight in 3ml LB medium containing appropriate antibiotic at 37˚C˚.
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Transfer the culture into a 1.5ml tube, centrifuge at 13,000 rpm for 1 minute, and discard the supernatant (2 times).
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Add 250 µL of SolutionⅠ to the pellet and mix well with vortex.
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Add 250 µL of SolutionⅡ and mix well by turning the tube upside down several times.
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Add 350 µL of SolutionⅢ and by turning the tube upside down several times.
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Centrifuge at 13,000 rpm for 5 minutes.
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Transfer the supernatant (approx. 850µL) to a mini prep column.
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Centrifuge at 13,000 rpm for 1 minute and discard the flow-through.
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Add 500 µL of SolutionⅣ.
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Centrifuge at 13,000 rpm for 1 minute and discard the flow-through.
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Add 700 µL of SolutionⅤ.
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Centrifuge at 13,000 rpm for 1 minute and discard the flow-through (2 times).
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Set the column on a new 1.5ml tube and add 100 µL of nuclease-free water.
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Centrifuge at 13,000 rpm for 1 minute and collect plasmid DNA.
Rapid check of the insert by colony cracking
Add 45 µL of cracking solution(3% w/v SDS ,50mM Tris -base ,pH12.6) into each tube.
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Suspend a small quantity of the colony in a tube using a sterilized toothpick.
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Incubate the tube at 65°C for 10 minutes.
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Add a drop of 10x Loading Buffer.
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Add equal volume of phenol/chloroform.
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Mix with vortex.
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Centrifuge at 13,000rpm for 3 minutes.
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Apply the supernatant to 1% agarose gel electrophoresis.
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Stain the gel in ethidium bromide solution for 10 minutes.
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Plasmid DNA can be detected by UV-illuminator as a band between genomic DNA band and low molecular size RNAs.
DNA purification and precipitation
PCR
SDS polyacrylamide gel electrophoresis (SDS-PAGE)
Isolation and purification of DNA bands
Ligation
Transformation
