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From 2013.igem.org

• purification of the limonene biobrick - BBa_24

1. Plasmid Volume

-1.1 - 40ul -1.2 - 40ul -1.3 - 46ul -1.4 - 39ul -1.5 - 41ul -1.2 - 32ul

1. measured DNA concentration using nanodrop

• DNA concentration (ng)

-1.1 - 131.8 -1.2 - 77.1 -1.3 - 56.8 -1.4 - 109 -1.5 - 129.1 -1.2 - 93.3

1. Restriction enzyme digest of the DNA samples

• enzymes: XbaI, PstI
• master mix:

Buffer - 14ul H2O - 88.2ul XbaI - 1.4ul PstI - 1.4ul Total volume - 105ul Each sample contained 5ul of DNA and 15ul of the master mix. The total amount of DNA in each sample was 200ng To calculate it we divided 200ng by the concentration of each sample The rest of the volume was made up with water to give a total of 5ul. The samples were them incubated in a water bath at 37C by an hour

• Agorose Gel preparation

-20ml of 5xTBE -80ml of water -0.8g of agarose

Boil for 3 minutes, 1 minute at a time. Let it cool and add 5ul of ethidium bromide. Pour and let set for 30 mins.

• Adding dye and and marker

Used orange g loading dye. -2ul to each sample

Used 1kb ladder, 5ul per 1 marker well.

• Run the gel