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From 2010.igem.org

Contents 1 PCR 2 ligation 3 transformation 4 isolation of plasmids 5 agarose gel

PCR

10 ul 5X Buffer 1 ul dNTP's 2 ul Primers (10 fold diluted) 1 ul Template 0.5 ul high fidelity polymerase 35.5 ul water

98°C 30s 98°C 30s 55°C 30s 72°C 60s 52°C 5min 4°C

35 cycles

ligation

10 ng storage plasmid 10 times more insert 1 ul ligase buffer (10x T4) fill up water to 10 ul

transformation

chemical:

isolation of plasmids

according to the protocol of NucleoSpin Plasmid (Macherey-Nagel)

agarose gel

1% agarose gel 0.5 g agarose 50 ml 1X TAE heat up with microwave, let cool down a bit 1 ul EtBr pour