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Team:ETHZ Basel/Biology/Protocols
From 2010.igem.org
(Difference between revisions)
(→PCR clean-up) |
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<br>10 ul 5X Buffer | <br>10 ul 5X Buffer | ||
<br>1 ul dNTP's | <br>1 ul dNTP's | ||
| - | <br>2 ul Primers (10 | + | <br>2 ul Primers (10 uM) |
<br>1 ul Template | <br>1 ul Template | ||
<br>0.5 ul high fidelity polymerase | <br>0.5 ul high fidelity polymerase | ||
<br>35.5 ul water | <br>35.5 ul water | ||
<br> | <br> | ||
| - | <br>98°C 30s | + | <br>initial denaturation 98°C 30s |
| - | <br>98°C 30s | + | <br>denaturation 98°C 30s |
| - | <br> | + | <br>touchdown annealing 58°C 30s, -0.2 °C/cycle until 55 °C reached |
| - | <br>72°C 60s | + | <br>polymerisation 72°C 60s/1kb -> 40 cycles |
| - | <br> | + | <br>final polymerisation 72°C 5min |
| - | <br>4°C | + | <br>hold 4°C |
| - | + | ||
| - | + | ||
== PCR clean-up == | == PCR clean-up == | ||
Revision as of 14:07, 6 August 2010
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
