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Team:ETHZ Basel/Biology/Protocols
From 2010.igem.org
(Difference between revisions)
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<br>35 cycles | <br>35 cycles | ||
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| + | == PCR clean-up == | ||
== ligation == | == ligation == | ||
Revision as of 13:46, 5 August 2010
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
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
