Team:Exeter/lab book/gibs/wk7
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- | <!-- | + | <!<p><b>**Tuesday 21.8.12**</b></p><br><p> |
+ | A digestion and gel of constructs made previously was run by Freddie. See lab book for 3 gene inducible plasmid for details. </p><br><p> | ||
+ | |||
+ | 2 step PCR of <i>wbbC</i> from genome</p><p> | ||
+ | <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/7" style="color:#1d1d1b"><u>Two step PCR</u></a></p><p> | ||
+ | Extra cycles at a lower annealing temperature were included due to primer overhangs not having DNA to anneal to in initial cycles</p><br><p> | ||
+ | |||
+ | PCR setup: </p><p> | ||
+ | 98°C for 30s</p><p> | ||
+ | 98°C for 10s, 55°C for 30s – X 5</p><p> | ||
+ | 98°C for 10s, 72°C for 30s – X 25</p><p> | ||
+ | 72°C for 5mins</p><p> | ||
+ | Hold temp. 4 °C</p><br><p> | ||
+ | |||
+ | Reaction 1: <i>fadR</i> control</p><p> | ||
+ | Reaction 2: genomic DNA</p><p> | ||
+ | DNA (10 ng/µl) – 2 µl</p><p> | ||
+ | Water – 34 µl</p><br><p> | ||
+ | |||
+ | 5 ml PCR reaction mix run on gel</p><br><p> | ||
+ | |||
+ | |||
+ | <b>**Wednesday 22.8.12**</b></p><br><p> | ||
+ | |||
+ | PCR of <i>wbbC</i> from genomic DNA</p><p> | ||
+ | <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/7" style="color:#1d1d1b"><u>Two step PCR</u></a></p><p> | ||
+ | DNA for attempt 1 same as before</p><br><p> | ||
+ | |||
+ | For 3/control (From <i>wbbC</i> (d) plasmid): </p><p> | ||
+ | DNA - 0.3 µl </p><p> | ||
+ | Water - 35.7 µl </p><br><p> | ||
+ | |||
+ | Ran gel – no joy</p><br><p> | ||
+ | |||
+ | |||
+ | <b>**Thursday 23.8.12**</b></p><br><p> | ||
+ | <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/7" style="color:#1d1d1b"><u>Two step PCR</u></a></p><p> | ||
+ | Nanodropped genomic DNA – got ~170 and 310 ng/µl</p><p> | ||
+ | Tried different concentrations</p><p> | ||
+ | Control DNA 0.3 µl, water 34.7 µl</p><p> | ||
+ | 1 – 1 µl gen. DNA, 34 µl water</p><p> | ||
+ | 2 – 0.8 µl gen. DNA, 34.2 µl water</p><p> | ||
+ | 3 – 0.5 µl gen. DNA, 34.5 µl water</p><p> | ||
+ | 4 – 0.1 µl gen. DNA, 34.9 µl water</p><br><p> | ||
+ | Gel ran– no luck</p><br><p> | ||
+ | |||
+ | Second go: </p><p> | ||
+ | <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/7" style="color:#1d1d1b"><u>Two step PCR</u></a></p><p> | ||
+ | Control – 0.3 µl DNA, water 34.7 µl</p><p> | ||
+ | 1 – 10 µl gen. DNA, 26 µl water</p><p> | ||
+ | 2 – 20 µl gen. DNA, 16 µl water</p><br><p> | ||
+ | |||
+ | <b>**Friday 24.8.12**</b></p><br><p> | ||
+ | |||
+ | PCR</p><p> | ||
+ | Control – same</p><p> | ||
+ | 1.5 µl of DMSO added</p><p> | ||
+ | Freddie then attempted a gradient PCR with serial dilutions of genomic DNA (see lab book for 3 gene inducible plasmid) </p> | ||
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Revision as of 17:29, 25 September 2012
Operon Construction: 20th - 24th August 2012 **Tuesday 21.8.12**A digestion and gel of constructs made previously was run by Freddie. See lab book for 3 gene inducible plasmid for details. 2 step PCR of wbbC from genome Extra cycles at a lower annealing temperature were included due to primer overhangs not having DNA to anneal to in initial cycles PCR setup: 98°C for 30s 98°C for 10s, 55°C for 30s – X 5 98°C for 10s, 72°C for 30s – X 25 72°C for 5mins Hold temp. 4 °C Reaction 1: fadR control Reaction 2: genomic DNA DNA (10 ng/µl) – 2 µl Water – 34 µl 5 ml PCR reaction mix run on gel **Wednesday 22.8.12** PCR of wbbC from genomic DNA DNA for attempt 1 same as before For 3/control (From wbbC (d) plasmid): DNA - 0.3 µl Water - 35.7 µl Ran gel – no joy **Thursday 23.8.12** Nanodropped genomic DNA – got ~170 and 310 ng/µl Tried different concentrations Control DNA 0.3 µl, water 34.7 µl 1 – 1 µl gen. DNA, 34 µl water 2 – 0.8 µl gen. DNA, 34.2 µl water 3 – 0.5 µl gen. DNA, 34.5 µl water 4 – 0.1 µl gen. DNA, 34.9 µl water Gel ran– no luck Second go: Control – 0.3 µl DNA, water 34.7 µl 1 – 10 µl gen. DNA, 26 µl water 2 – 20 µl gen. DNA, 16 µl water **Friday 24.8.12** PCR Control – same 1.5 µl of DMSO added Freddie then attempted a gradient PCR with serial dilutions of genomic DNA (see lab book for 3 gene inducible plasmid) |