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From 2010.igem.org

PCR

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

initial denaturation 98°C 30s
denaturation 98°C 30s
touchdown annealing 58°C 30s, -0.2 °C/cycle until 55 °C reached
polymerisation 72°C 60s/1kb -> 40 cycles
final polymerisation 72°C 5min
hold 4°C

PCR clean-up

according to the protocol of GenElute PCR Clean-Up Kit (Sigma-Aldrich)

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