Team:Macquarie Australia/assembly

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3A Assembly

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Restriction Digest

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Master mix reactions were prepared as per table below.

Plasmid Backbone Master Mix	Part A Master Mix	Part B Master Mix
5µl CutSmart Buffer	5µl CutSmart Buffer	5µl CutSmart Buffer
0.5µl EcoRI-HF	0.5µl EcoRI-HF	0.5µl XbaI
0.5µl PstI	0.5µl SpeI	0.5µl PstI
18µl Deionised Water	18.5µl Deionised Water	18.5µl Deionised Water

Digest reactions were then run according to table below at 37^oC for 30 minutes followed by 80℃ for 20 minutes.

Digest reaction Plasmid Backbone	Digest reaction Part A	Digest reaction Part B
4µl linerarized plasmid backbone (25ng/µl for 100ng total)	4µl Part A (25ng/µl for 100ng total)	4µl Part B (25ng/µl for 100ng total)
4µl of Plasmid Backbone Master Mix	4µl Part A Master Mix	4µl Part B Master Mix

Ligation

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2µl of digested Plasmid Backbone an equimolar amount of digested Part A (< 3µl) and an equimolar amount of digested Part B (< 3µl) were added to 1µl T4 DNA ligase buffer and 0.5µl of T4 DNA ligase. Water was added to bring the total to 10µl. Ligation was performed for 30 minutes at 16^oC followed by a further period of 20 minutes at 80^oC.

Transformation

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1. Obtain competent cells from -80^oC.
2. Defrost gently on ice. 100µl is sufficient for 2 transformations.
3. Add 1-10µl of plasmid DNA/ ligation mix to each tube. Incubate on ice for 30 minutes.
4. Heat shock in 42^oC water bath for 30 seconds, then back on ice for 2 minutes.
5. Add 200µl of SOC media to each tube, and incubate in the 37^oC shaker for 1 hour.
6. For each tube of cells, spread 50µl onto one LB plate with appropriate antibiotic, and 100µl onto a second plate, using aseptic technique. Place your plate upside-down in the 37^oC incubator.