Team:Exeter/lab book/proto/11

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Protocol 11


Protocols \| Single Gene Plasmids and Enzyme Characterisation \| Showcasing Polysaccharide Production \| The 3-Gene Inducible Plasmid Operon Construction \| Glycobase

Preparation of IDTE Buffer and IDT Re-Suspension
Spin down IDT synthesised gene for 10 seconds to ensure the DNA is at the bottom of the tube. In a Duran bottle, add Tris (11.31g) to MilliQ H₂O (40mL) and using a stir bar and magnetic stirrer, mix the solution thoroughly for 1 minute. Add 1M NaOH drop-wise until all the Tris is dissolved and the pH reaches approximately pH 9. Top up the solution to 50mL using MilliQ H₂O to make a 10mM Tris solution and autoclave for 20 minutes. In a separate Duran bottle, add EDTA (1.21g) to MilliQ H₂O (60mL) and using a stir bar and magnetic stirrer, mix the solution thoroughly for 1 minute. Add 1M HCl drop-wise until the pH drops to pH 7.5. Top up the solution to 100mL using MilliQ H₂O to make a 0.1mM EDTA solution, and then autoclave for 20 minutes. Finally, add appropriate volumes of Tris and EDTA solutions to make a 50mL solution with 10mM Tris, 0.1mM EDTA. Use this final IDTE solution for re-suspension of all synthesised genes. Add 40µL of IDTE buffer to each synthesised gene, held between pH 7.5-8.0, to reach an approximate concentration of 50ng/µL stock solution. Vortex for 20 seconds. Leave to incubate at room temperature for 30 minutes. Centrifuge for 1 minute at 16’100rcf. To dilute each synthesised gene for transformation, add 2µL of the stock solution to 999µL MilliQ H₂O to reach an approximate concentration of 0.1ng/µL. Centrifuge briefly to spin down any liquid. Use between 1-5µL of this diluted stock solution for transformation. Store remaining hydrated DNA stock solution from the IDT re-suspension at -20^°C until further use required.

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