Team:Exeter/lab book/gibs/wk5

Operon Construction  |  Glycobase

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3A BioBrick assembly of RBS-wbbC(d) (BBa_B0034 + BBa_K764005)

Upstream

RBS 3 (BBa_B0034) plasmid DNA - 8.1 µl

Water - 14.4 µl

Downstream wbbC(d)

DNA - 2.8 µl

Water - 19.7 µl

Destination plasmid pSB1C3 (chloramphenicol resistant) Digestion

Ligation

Upstream: 2 µl

Downstream: 2 µl

MilliQ water 2.5

23 °C (room temp.) incubation for 1 hour

20 minute heat kill 80 °C

**Wednesday 8.8.12**

Transformed RBS-wbbC(d) (BBa_B0034 + BBa_K764005)

2 µl into 25 µl competent cells

50 µl and 150 µl LB(chlor) spread plates made

**Thursday 9.8.12**

Transferred RBS-wbbC(d) (BBa_B0034 + BBa_K764005) transformed colonies to LB(chlor) medium

**Friday 10.8.12**

RBS-wbbC(d) transformed cells broth tubes centrifuged for 5 minutes, 3901 rcf, 4 °C

Supernatant discarded

Miniprep according toFermentas protocol

Step 1045µl milli-Q water added in place of elution buffer, followed by another 5 µl after incubation and centrifugation