Team:Exeter/lab book/gibs/wk7

Operon Construction  |  Glycobase

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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

Two step PCR

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

Two step PCR

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**

Two step PCR

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:

Two step PCR

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)