"

From 2010.igem.org

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

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