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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{{ETHZ_Basel10}}
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{{ETHZ_Basel10_Biology}}
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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 uM)
-
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>initial denaturation 98°C 30s
-
98°C 30s
+
<br>denaturation 98°C 30s
-
55°C 30s
+
<br>touchdown annealing 58°C 30s, -0.2 °C/cycle until 55 °C reached
-
72°C 60s
+
<br>polymerisation 72°C 60s/1kb -> 40 cycles
-
52°C 5min
+
<br>final polymerisation 72°C 5min
-
4°C
+
<br>hold 4°C
-
 
+
-
35 cycles
+
 +
== PCR clean-up ==
 +
according to the protocol of GenElute PCR Clean-Up Kit (Sigma-Aldrich)
== ligation ==
== ligation ==
-
10 ng storage plasmid
+
<br>10 ng storage plasmid
-
10 times more insert
+
<br>10 times more insert
-
1 ul ligase buffer (10x T4)
+
<br>1 ul ligase buffer (10x T4)
-
fill up water to 10 ul
+
<br>fill up water to 10 ul
== transformation ==
== transformation ==
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== agarose gel ==
== agarose gel ==
-
1% agarose gel
+
<br>1% agarose gel
-
0.5 g agarose
+
<br>0.5 g agarose
-
50 ml 1X TAE
+
<br>50 ml 1X TAE
-
heat up with microwave, let cool down a bit
+
<br>heat up with microwave, let cool down a bit
-
1 ul EtBr
+
<br>1 ul EtBr
-
pour
+
<br>pour

Latest revision as of 11:45, 12 October 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

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