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Team:ETHZ Basel/Biology/Protocols
From 2010.igem.org
(Difference between revisions)
(New page: == 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 ...) |
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== PCR == | == PCR == | ||
| - | 10 ul 5X Buffer | + | <br>10 ul 5X Buffer |
| - | 1 ul dNTP's | + | <br>1 ul dNTP's |
| - | 2 ul Primers (10 fold diluted) | + | <br>2 ul Primers (10 fold diluted) |
| - | 1 ul Template | + | <br>1 ul Template |
| - | 0.5 ul high fidelity polymerase | + | <br>0.5 ul high fidelity polymerase |
| - | 35.5 ul water | + | <br>35.5 ul water |
| - | + | <br> | |
| - | 98°C 30s | + | <br>98°C 30s |
| - | 98°C 30s | + | <br>98°C 30s |
| - | 55°C 30s | + | <br>55°C 30s |
| - | 72°C 60s | + | <br>72°C 60s |
| - | 52°C 5min | + | <br>52°C 5min |
| - | 4°C | + | <br>4°C |
| - | + | <br> | |
| - | 35 cycles | + | <br>35 cycles |
| - | + | ||
== ligation == | == ligation == | ||
Revision as of 13:36, 5 August 2010
Contents |
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
