Team:Carnegie Mellon/Met-Protocols

From 2012.igem.org

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<b> Kit Protocols</b></h2>
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<tr> <td width = "25"> 1. </td><td width = "200"> <a href="http://www.zymoresearch.com/downloads/dl/file/id/166/t3001i.pdf">Z-Competent E. Coli Transformation Kit</a> for making competent cells for easy transformation. Modified to incubate cells on ice for 1 hour during transformation</td></tr>
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<tr><td>2. </td><td> <a href ="http://www.neb.com/nebecomm/ManualFiles/manualE0553.pdf"> Phusion High Fidelity PCR Kit </a> for amplifying our inserts and cassettes. </td></tr>
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<tr><td>3. </td><td> Wash 1mL induced cells once with PBS and resuspend in 1mL M9 media.</td></tr>
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<tr><td>4. </td><td title="To increase DFHBI fluorescence"> Add 1µL of 50nM Mg2+ to the tubes (from MgCl2 from PCR kit)</td></tr>
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<tr><td>5. </td><td>Aliquot (100ul/200ul) of cells into desired wells of a 96-well plate.</td></tr>
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<tr><td>6. </td><td>Add various doses of DFHBI to the wells, followed by adding the desired doses of MG</td></tr>
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<tr><td>7. </td><td>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. </td></tr>
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<b>Dosage Curve</b></h2>
<b>Dosage Curve</b></h2>
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<b>Cloning Protocol</b></h2>
<b>Cloning Protocol</b></h2>
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<tr><td>3. </td><td>Purify and clean DNA using kit. [Zymo Research] </td></tr>
<tr><td>3. </td><td>Purify and clean DNA using kit. [Zymo Research] </td></tr>
<tr><td>4. </td><td>Measure vector/insert concentration. [Nanodrop] </td></tr>
<tr><td>4. </td><td>Measure vector/insert concentration. [Nanodrop] </td></tr>
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<tr><td>5. </td><td>Divide the concentration by the length and prepare the ligation reagents accordingly, with  </td></tr>
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<tr><td>5. </td><td>Divide the concentration by the length of the sequence and calculate ligation ratios of 1 vector to 3 insert. Mix the ratios according to the calculations, including T4 buffer and ligase. </td></tr>
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<tr><td>6. </td><td>Leave ligation products at room temperature for 1 hour. </td></tr>
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<tr><td>7. </td><td>Transform ligation products into competent cells using appropriate protocol. Incubate on ice for 1 hour if using Zymo competent cells </td></tr>
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<tr><td>8. </td><td>Plate cells and incubate at 37 degrees overnight. Check for colonies the next day </td></tr>
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Revision as of 15:27, 21 September 2012

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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.

Kit Protocols

1. Z-Competent E. Coli Transformation Kit for making competent cells for easy transformation. Modified to incubate cells on ice for 1 hour during transformation
2. Phusion High Fidelity PCR Kit for amplifying our inserts and cassettes.
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.

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 of the sequence and calculate ligation ratios of 1 vector to 3 insert. Mix the ratios according to the calculations, including T4 buffer and ligase.
6. Leave ligation products at room temperature for 1 hour.
7. Transform ligation products into competent cells using appropriate protocol. Incubate on ice for 1 hour if using Zymo competent cells
8. Plate cells and incubate at 37 degrees overnight. Check for colonies the next day

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