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Making agar plates

Pouring normal LB + agar plates (with optional antibiotic)

Preparing the LB+agar powder

Mix the contents below to prepare the agar powder required to make 10L of agar solution

• 100gr tryptoptan
• 50gr yeast extract
• 50gr NaCl
• 150 gr agar

Mix the content very well before using the mixed powder

Preparing the LB+agar solution

To prepare 1L of LB+agar solution:

• Put 500 ml of distilled water into a measuring cylinder then transfer to a clean 1000ml flask

• Put 400ml of distilled water to a measuring cylinder and then transfer this to another 1000ml flask. To this flask add 35 gr of LB+agar powder. After mixing sufficiently transfer the LB+agar solution into the measuring cylinder and add distilled water until a total volume of 500ml has been reached.

You should now have two 1000ml flasks - one conatining 500ml of distilled water and the other containing 500ml of LB+agar solution

• Add 10ul of antifoam to LB+agar flask

• Seal the flask with some tin foil and place autoclaving tape on it. Make sure the flasks are correctly labelled with date, substance within flask and lab.

• Put the flasks in the autoclaver for half an hour

• Once the flasks have had time to cool for a few mins, carefully place the flask with LB+agar into 55C-60C water bath.

• Also, put the flask containing distilled water into the sink to cool it down - only once it is cool enough to transfer. 'This means that the water flask should cool down more rapidly than the LB+agar flask.

• Once the LB+agar flask has got to the temperature where it is still very warm but not unbearable (you should be able to put your hand on the base without burning yourself), pour the distilled water from the other flask into it. Make sure that the bunsen flame is used - this step must be aseptic

• Swirl a few times and then, using aseptic technique, pour all the contents from the LB flask back into the currently empty 'distilled water' flask. Seal with foil.

By placing all of the content into the LB flask and then pouring it all back into the distilled water flask, you are sufficiently mixing the solutions. And because the distilled water flask has been cooling more rapidly than the LB flask, the solution will be stored in the cooler flask before being poured into plates - this is an optimum condition.

• If necessary, add the antibiotics to LB solution flask.

You are now ready to pour the plates - make sure that you do this immediately otherwise the LB+agar will cool and solidify in the flask

Pouring the plates

• Pour the plates using roughly 25ml of the solution for each Petri dish using the aseptic technique
• Leave the dishes for 30 minutes in room temperature
• Put them into 42C incubator with their lids off to dry them off
• If you are not using the dishes straight away leave them on the bench for a day before placing them to the fridge

Making up starch plates

Starch plates are useful for determining whether bacteria have become transformed by DNA if that plasmid happens to render amyE (a gene encoding the enzyme Amylase which breaks down starch) inactive. For example, the GFP-rrnb vector plasmid causes amyE knock-out in Bacillus subtilis.

To make up LB + agar + starch plates, you need to follow the normal protocol for making 1 litre of LB+agar solution (see above) except you will need to add 10g of starch powder to the 500ml LB+agar solution flask before autoclaving. 1 gram of starch powder to 100ml of LB is the correct ratio.