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Team:Carnegie Mellon/Met-Protocols
From 2012.igem.org
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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> | <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> | ||
<tr><td>3. </td><td> <a href = "http://www.qiagen.com/literature/render.aspx?id=201794">Qiagen Mini-prep Kit </a> for isolating our plasmids from the cells, to be transformed into expression strains. </td></tr> | <tr><td>3. </td><td> <a href = "http://www.qiagen.com/literature/render.aspx?id=201794">Qiagen Mini-prep Kit </a> for isolating our plasmids from the cells, to be transformed into expression strains. </td></tr> | ||
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</table> | </table> | ||
</p> | </p> | ||
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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> | <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> | ||
<tr><td>8. </td><td>Plate cells and incubate at 37 degrees overnight. Check for colonies the next day </td></tr> | <tr><td>8. </td><td>Plate cells and incubate at 37 degrees overnight. Check for colonies the next day </td></tr> | ||
| + | </table> | ||
| + | </p> | ||
| + | |||
| + | <p><h2 id = "section1-5"> | ||
| + | <b>Gel Protocol</b></h2> | ||
| + | |||
| + | <table> | ||
| + | <tr> <td width = "25"> 1. </td><td width = "200"For 7x7 cm, 0.5cm thick gel, with 1.75% agarose Concentration. </td></tr> | ||
| + | <tr><td>2. </td><td> Add 0.35 grams of agarose to 20ml of 1x buffer (TBE) | ||
| + | Note: Place mark on container to keep track of liquid level using marker, so water can be added to bring the liquid back to original level </td></tr> | ||
| + | <tr><td>3. </td><td>Place gel solution in microwave. Use low/medium, set timer for 5 minutes. Stop the oven every 30 seconds and swirl gently to suspend undissolved agarose. </td></tr> | ||
| + | <tr><td>4. </td><td>Once dissolved, set aside to cool (~60 degrees). Once solution is warm to touch, add 0.5ug/ml ethidium bromide. [1ul of 10mg/ml]</td></tr> | ||
| + | <tr><td>5. </td><td>Pour into casting tray, remember to add in comb at the cathode side (black). Gel will solidify in ~10 mins</td></tr> | ||
| + | <tr><td>6. </td><td> Remove comb for solidified gel, remove casting gates and submerge gel beneath 2 to 6mm of 1x buffer. </td></tr> | ||
| + | <tr><td>7. </td><td> Mix loading dye with PCR/digested products, load mixture into wells, together with 10ul of ladder </td></tr> | ||
| + | <tr><td>8. </td><td>Run gel at 75V for ~1 hour. Adjust voltage accordingly if require faster or more distinct gels.</td></tr> | ||
</table> | </table> | ||
</p> | </p> | ||
Revision as of 15:43, 21 September 2012
Protocols
Overview
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. | Qiagen Mini-prep Kit for isolating our plasmids from the cells, to be transformed into expression strains. |
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. [NEB] |
| 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 |
Gel Protocol
| 1. | |
| 2. | Add 0.35 grams of agarose to 20ml of 1x buffer (TBE) Note: Place mark on container to keep track of liquid level using marker, so water can be added to bring the liquid back to original level |
| 3. | Place gel solution in microwave. Use low/medium, set timer for 5 minutes. Stop the oven every 30 seconds and swirl gently to suspend undissolved agarose. |
| 4. | Once dissolved, set aside to cool (~60 degrees). Once solution is warm to touch, add 0.5ug/ml ethidium bromide. [1ul of 10mg/ml] |
| 5. | Pour into casting tray, remember to add in comb at the cathode side (black). Gel will solidify in ~10 mins |
| 6. | Remove comb for solidified gel, remove casting gates and submerge gel beneath 2 to 6mm of 1x buffer. |
| 7. | Mix loading dye with PCR/digested products, load mixture into wells, together with 10ul of ladder |
| 8. | Run gel at 75V for ~1 hour. Adjust voltage accordingly if require faster or more distinct gels. |
