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Materials and Methodology

Preparation of LB Medium and 1.5% Agar


As the standard nutrient media for propagation of E. coli an LB rich medium was prepared
by mixing, in 1 litre of water, 10 g of tryptone, 5 g of yeast extract and 10 g of NaCl. The solutions
pH was adjusted to 7.5. The solution was then distributed into various containers and autoclaved.
For growth of the bacteria in plates an LB agar was prepared with 1.5% agar by adding
1.5g of agar per 100 ml of LB solution. It was then autoclaved and stored. When ready to use the
agar was melted in the microwave and then stirred with a magnetic stirrer while it cooled down.
Once hand-hot the appropriate quantity of Ampicillin, Streptomycin, IPTG or X-GAL (as required)
were added and aproximately 20 ml poured into each petri dish which were left in a microbial
safety cabinet to set.

Growth of E. coli XL1-Blue cells


E. coli XL1-Blue cells were grown on their own, to provide competent cells, with ligation
mixes or with previously prepared plasmids. They were grown either in liquid media in 5, 50 or
100 ml of LB solution or on agar plates with the suitable antibiotic (tetracyclin in this case) at
37oC.

Preparation of Ampicillin, Streptomycin, X-GAL and IPTG


The antibiotics were used for colony selection, i.e. colonies which had uptaken the
pBluescript vector with the mutated insert (white colonies) over the colonies with an empty vector
(blue colonies) and to assure the growth of a single strain of bacteria. Colour selection is due to
inactivation of the -galactosidase gene in the vector by the insert. IPTG (25 mg.ml -1 stock
solution at 1000x working concentration) induces the synthesis of -galactosidase and
X-GAL (25 mg.ml-1 stock solution at 1000x working concentration) of one of its substrates were
added to the hand-hot 1.5% agar solution before pouring it into plates.
Streptomycin (50 mg.ml -1 stock solution at 1000x working concentration) and Ampicillin
(100 mg.ml-1 stock solution at 1000x working concentration) were added to the liquid media when
appropriate or to the cooled 1.5% agar.

Preparation of competent cells


A few colonies were picked from the E. coli XL1-Blue culture and flicked into 50 ml of LB
solution. The mixture was shaken overnight at 180 rpm and 37 oC and from this culture 1 ml was
sub-cultured into 50 ml of LB and left in the shaker (at 180 rpm and 37 oC) until it achieved midexponential phase of growth (OD 600nm= 0.4 - 0.6). The culture was equally distributed into two
sterile Falcon tubes and centrifuged at 7000 rpm for 10 minutes at 4 oC. The supernatant of each
tube was discarded and the pellet resuspended in 10 ml of ice cold 50 mM CaCl 2 and left on ice
for an hour. The samples were centrifuged again using the same procedure as before and the
resulting pellets were resuspended this time in 2 ml of CaCl 2, each tube, and left to rest on ice
until ready to be used.

Ligations
Ligation reactions between vectors and inserts were set up by adding 1l of freshly
prepared 7.5 mM ATP, 1.5 l of T4 DNA Ligase buffer, insert and vector samples (combined
volume of 11 l) and lastly 1.5 l of T4 DNA Ligase, to give a total volume of 15 l. Reaction
mixtures were incubated at 12oC for at least 16 hours.

Transformation into competent cells


To transform the mutated gene into the competent E. coli XL1-Blue cells, to each ligation
mixture or previously prepared plasmids 200 l of competent cells were added in microcentrifuge
tubes, vortexed and kept on ice for 30 minutes. The cells were then heat-shocked in a 42 oC bath
for 1 minute and relocated to ice for 2 5 minutes. 0.8 ml of LB solution was added to the cell
mixture (total volume of 1 ml) which was then left to incubate for 30 minutes at 37 oC to revive the
cells. 200 l of each sample were spread onto agar plates.

Plasmid preparation kit


Used to extract the plasmid DNA from the vectors.
Selected colonies were picked and grown overnight in 5 ml of LB with 5 l of ampicillin at
37oC. The resulting culture was distributed in 1.5 l volumes

Gel agarose electrophoresis


Gel extraction kit
Restriction enzymes digestions
Dephosphorylation
Primer design

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