Team:Exeter/lab book/novpol/wk5

Operon Construction  |  Glycobase

-

-

-

-

-

-

-

-

-

Monday 6th August - Annealed oligomers together of ompA to make the signal peptide and ligated to plasmid.

Extracted DNA 2.0 order of HAS, cyclodextrin and wbiP.

Using:
( Amount of vector x size of insert ) / size of vector 25ng vector x 0.1 kb insert / 2 kb vector = 1.25ng

Since our T4 ligase ligation protocol we wanted equimolar amounts of insert: vector

Dissolved oligomers in annealing buffer (10mM tris-Cl, pH 8.0, 1M EDTA and 50mM NaCl). 223ul of annealing buffer was added to fragment 1 and 96ul was added to fragment 2. 25ul of each appropriate oligomer pair was mixed into a micro-eppendorf. The PCR machine was programmed to heat to 95°C for two minutes and then cool to 30°C by decreasing by 0.5°C every 30 seconds. Annealed ompA was then cooled to 4°C.

Used plasmid digest from Friday to ligate to ompA. 2ul of digested plasmid (25ng) was added to an equimolar amount of the fragment and to 1ul T4 DNA ligase buffer and 0.5ul T4 DNA ligase. Water was added to make up the volume to 10ul. Left to ligate at room temperature for 20 minutes and then placed in the fridge for overnight.

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 13000rms and transfer 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 7th August - Transformed ompA, HAS, cyclodextrin and wbiP into E. coli. Also streak plated SacB which was sent on a swab sample from the biobrick registry.

(see previous transformation procedures)

Wednesday 8th August - Transferred genes to liquid broth to grow overnight.

(see previous transferral to liquid broth procedures).

Thursday 9th August - Mini prep of ompA, HAS, cyclodextrin, SacB and wbiP. 3A assembly of HAS, cyclodextrin, and wbiP to the terminator and of SacB and cyclodextrin to ompA.

(For concentrations of ompA, HAS, cyclodextrin and wbiP see results. For mini prep procedure see previous mini prep procedures).