Team:Exeter/lab book/novpol/wk5

Operon Construction  |  Glycobase

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**Monday 06/08/12**

Annealed oligomerstogether of ompA to make the signal peptide using equimolar amounts of insert: vector. Ligated ompA to chloramphenicol plasmid using 3A assembly guidelines for ligation. Extracted DNA 2.0 order of HAS, cyclodextran and wbiP.

The DNA 2.0 genes were extracted by removing filter paper and placing in a petri dish. Added 100ul of IDTE buffer (10mM Tris, 0.5mM EDTA, pH 7.5) to the filter papers and allowed them to absorb the buffer. The genes were incubated at room temperature for two minutes. Punctured the bottom of a medium sized eppendorf and placed inside a large eppendorf. Used tweezers to roll the filter paper and added to the medium eppendorf. Spun in the centrifuge for 1 minute at full speed and transferred the DNA from the filter paper into the large eppendorf so the medium sized eppendorf and filter paper could be discarded. Supernatant should contain approximately (20ng/ul).

The DNA was placed in the freezer until the next day.

**Tuesday 07/08/12**

Transformed ompA, HAS, cyclodextran and wbiP into E coli. OmpA was added to a chloramphenicol plate, HAS, cyclodextran and wbiP to kanamycin plates. Streak plated SacB which was sent on a swab sample from the BioBrick registry.

**Wednesday 08/08/12**

Transferred genes to liquid broth containing their correct selection antibiotic, to grow overnight.

**Thursday 09/08/12**

Mini prep of ompA, HAS, cyclodextran, SacB and wbiP. 3A assembly performed of HAS, cyclodextran, and wbiP to the terminator and of SacB and cyclodextran to ompA.