Team:Exeter/lab book/novpol/wk7

Operon Construction  |  Glycobase

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**Monday 20/08/12**

Went through all previous constructs and mini-peps to figure out what had worked and what hadn’t. Performed a digest and ran them all on a gel using electrophoresis.

None of the constructs had worked correctly as they only single banded on the gel.

**Tuesday 21/08/12**

Digestion and ligation of the original HAS, cyclodextran and sacB onto a terminator, into digested tetracycline plasmids using 3A Assembly. The ligated BioBricks were transformed onto plates that contained the tetracycline antibiotic. ompA oligomers were annealed together in the PCR machine. The annealed ompA was then ligated into a chloramphenicol plasmid and transformed also.

**Wednesday 22/08/12**

None of the colonies transformed yesterday had grown overnight. Repeated the digestion and ligation procedure from yesterday again but left them to digest in the incubator for an hour and left them to ligate at room temperature for two hours. Transformed and left overnight again.

**Thursday 23/08/12**

The colonies had grown overnight on the plates except for ompA. Transferred the constructs to liquid broth and placed in a shaking incubator overnight. The remaining ligation mix of OmpA (7ul) from previously was added to cells and transformed.

**Friday 24/08/12**

Mini prep of the constructs HAS-terminator, cyclo-terminator and sacB-terminator. Confirmed nucleic acid present using the nanodrop machine. Digested the fragments using the 3A Assembly protocol and ran on a gel for electrophoresis. Several bands obtained but the ligation had not worked.

ompA had successfully grown on the plate so sealed the plate and placed in the fridge.