Team:Carnegie Mellon/Met-Protocols

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Protocols

Overview

Our protocols are based on standard lab protocols used in the lab of our advisers unless otherwise stated. These protocols were subsequently modified if necessary based on our experience. A knowledge of basic lab techniques such as pippeting, plating, etc. are assumed and not covered as there are pretty comprehensive tutorials on the web for those.

Dosage Curve

1. First culture a fresh batch of cells using 1:100 of overnight culture of promoter construct. Culture for around 2 hours or until the solution is lightly turbid.
2. Induce cells by adding 1ul of 1mM IPTG.
3. Wash 1mL induced cells once with PBS and resuspend in 1mL M9 media.
4. Add 1µL of 50nM Mg2+ to the tubes (from MgCl2 from PCR kit)
5. Aliquot (100ul/200ul) of cells into desired wells of a 96-well plate.
6. Add various doses of DFHBI to the wells, followed by adding the desired doses of MG
7. Plate read with plate reader (Tecan Safire II). First find cell density using OD600. Then use Excitation/Emission of 469/501 for Spinach and 635/660 for Malachite-green.

Cloning Protocol

1. Start digestion of vector and insert DNA using desired restriction enzyme manufacturer protocol.
2. After 2 hours, add Phosphatase [CIP] to insert to prevent self-ligation
3. Purify and clean DNA using kit. [Zymo Research]
4. Measure vector/insert concentration. [Nanodrop]
5. Divide the concentration by the length and prepare the ligation reagents accordingly, with

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