Team:Hong Kong-CUHK/lab2.html
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<p><strong><a name="a1.1" id="a1.1"></a>1.1 Gene Amplification with PCR</strong></p> | <p><strong><a name="a1.1" id="a1.1"></a>1.1 Gene Amplification with PCR</strong></p> | ||
- | < | + | <p>1. Put the reaction tubes on ice.<br /> |
- | + | 2. Add the following components into each reaction tube:</p> | |
- | + | ||
- | + | ||
- | + | ||
<ul> | <ul> | ||
<li>5x Phusion HF Buffer* (1X final concentration is recommended)</li> | <li>5x Phusion HF Buffer* (1X final concentration is recommended)</li> | ||
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<li>Phusion DNA Polymerase (0.02 U/μl final concentration is recommended)</li> | <li>Phusion DNA Polymerase (0.02 U/μl final concentration is recommended)</li> | ||
</ul> | </ul> | ||
- | < | + | <p>3. Mix well and run the following program:</p> |
- | + | ||
- | + | ||
<p> </p> | <p> </p> | ||
<p>2-step PCR Cycling Program<br /> | <p>2-step PCR Cycling Program<br /> | ||
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<p>Hold:<br /> | <p>Hold:<br /> | ||
4°C</p> | 4°C</p> | ||
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<p><strong><a name="a1.2" id="a1.2"></a>1.2 PCR Purification*</strong></p> | <p><strong><a name="a1.2" id="a1.2"></a>1.2 PCR Purification*</strong></p> | ||
- | < | + | <ol> |
<li>Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix.</li> | <li>Add 5 volumes of Buffer PB to 1 volume of the PCR sample and mix.</li> | ||
<li>If pH indicator I has been added to Buffer PB, check that the color of the mixture is yellow. If the color of the mixture is orange or violet, add 10 µl of 3 M sodium acetate (pH 5.0), and mix. The color of the mixture will turn to yellow.</li> | <li>If pH indicator I has been added to Buffer PB, check that the color of the mixture is yellow. If the color of the mixture is orange or violet, add 10 µl of 3 M sodium acetate (pH 5.0), and mix. The color of the mixture will turn to yellow.</li> | ||
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<li>To elute DNA, add 50 μl Buffer EB (10 mM Tris-Cl, pH 8.5) or water (pH 7.0–8.5) to the center of the QIAquick membrane and centrifuge the column for 1 min. Alternatively, for increased DNA concentration, add 30 μl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge.</li> | <li>To elute DNA, add 50 μl Buffer EB (10 mM Tris-Cl, pH 8.5) or water (pH 7.0–8.5) to the center of the QIAquick membrane and centrifuge the column for 1 min. Alternatively, for increased DNA concentration, add 30 μl elution buffer to the center of the QIAquick membrane, let the column stand for 1 min, and then centrifuge.</li> | ||
<li>If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.</li> | <li>If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.</li> | ||
- | </ | + | </ol> |
<p>IMPORTANT: Ensure that the elution buffer is dispensed directly onto the QIAquick membrane for complete elution of bound DNA. The average eluate volume is 48 µl from 50 µl elution buffer volume, and 28 µl from 30 µl elution buffer.</p> | <p>IMPORTANT: Ensure that the elution buffer is dispensed directly onto the QIAquick membrane for complete elution of bound DNA. The average eluate volume is 48 µl from 50 µl elution buffer volume, and 28 µl from 30 µl elution buffer.</p> | ||
<p>*Protocol adopted from QIAquick PCR purification kit protocol</p> | <p>*Protocol adopted from QIAquick PCR purification kit protocol</p> | ||
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<p><strong><a name="a1.3" id="a1.3"></a>1.3 Overlapping PCR</strong></p> | <p><strong><a name="a1.3" id="a1.3"></a>1.3 Overlapping PCR</strong></p> | ||
- | < | + | <p>1. Put the reaction tubes on ice.<br /> |
- | + | 2. Add the following components into each reaction tube:</p> | |
- | + | ||
- | + | ||
- | + | ||
<ul> | <ul> | ||
<li>5x Phusion HF Buffer* (1X final concentration is recommended)</li> | <li>5x Phusion HF Buffer* (1X final concentration is recommended)</li> | ||
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<li>Phusion DNA Polymerase (0.02 U/μl final concentration is recommended)</li> | <li>Phusion DNA Polymerase (0.02 U/μl final concentration is recommended)</li> | ||
</ul> | </ul> | ||
- | < | + | <p>3. Mix well and run the following program:</p> |
- | + | ||
- | + | ||
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<p>2-step PCR Cycling Program<br /> | <p>2-step PCR Cycling Program<br /> | ||
Initial denaturation:<br /> | Initial denaturation:<br /> | ||
- | + | 98°C 30 seconds</p> | |
<p>25–35 cycles:<br /> | <p>25–35 cycles:<br /> | ||
98°C 10 seconds (denaturation)<br /> | 98°C 10 seconds (denaturation)<br /> | ||
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<p>Hold:<br /> | <p>Hold:<br /> | ||
4°C</p> | 4°C</p> | ||
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<p><strong><a name="a1.4" id="a1.4"></a>1.4 Agarose Gel Electrophoresis</strong></p> | <p><strong><a name="a1.4" id="a1.4"></a>1.4 Agarose Gel Electrophoresis</strong></p> | ||
- | < | + | <p><strong>A. </strong>Cast gel</p> |
- | + | <p>1. Dissolve 0.55 g agarose into 55 ml 0.5X TBE buffer.<br /> | |
- | + | 2. | |
- | + | Microwave (high power, 800W) for 1 min.<br /> | |
- | + | 3. | |
- | + | Cool it down using running water for 1 min.<br /> | |
- | + | 4. | |
- | + | Add 1 μl GelRed<br /> | |
- | < | + | 5. |
- | < | + | Pour the solution to tightened tank with gates and gel comb and allow it to solidify.<br /> |
- | + | 6. | |
- | + | Transfer the gel to gel tank once it becomes solid.</p> | |
- | + | <p><strong>B. </strong>Run gel</p> | |
- | + | <p>1. Orient the gel with wells facing the black negative electrode. Check if the gel is covered by TBE buffer in the tank. If not, add TBE buffer to cover it to about 1mm.<br /> | |
- | + | 2. | |
- | + | Mix loading dye and the insert/plasmid before adding to the wells. For example, if the DNA we have got is 45μl, and the loading dye we have got is 10X, then add 5μl of loading dye to the samples. Mixture should be in blue.<br /> | |
- | + | 3. | |
- | <p><a href="#top">Back To Top</a></p> | + | To run the gel, add all samples to the wells of gel. Then add 1kb DNA ladder to a separate well. 1μl should be enough for detection under UV.<br /> |
+ | 4. | ||
+ | Connect the electrodes to the power supply with correct colour. Set the power supply to 120V. Check if there are bubbles on the negative electrodes.<br /> | ||
+ | 5. | ||
+ | Allow it to run for about 40 - 60 min. To avoid running the band off the gel, the yellow band (position of the smallest fragments) should stay on the gel.</p> | ||
+ | <p><a href="#top" style="text-decoration : underline; color: #090">Back To Top</a></p> | ||
<p><strong><a name="a1.5" id="a1.5"></a>1.5 Preparation of Competent Cells</strong></p> | <p><strong><a name="a1.5" id="a1.5"></a>1.5 Preparation of Competent Cells</strong></p> | ||
<p><strong>A. </strong>Preparation of detergent-free glassware and media</p> | <p><strong>A. </strong>Preparation of detergent-free glassware and media</p> | ||
- | < | + | <p>1. Autoclave glassware filled with 3/4 dd-H2O to remove most residual detergent.<br /> |
- | + | 2. | |
- | + | Autoclave media and buffers in detergent-free glassware</p> | |
- | + | ||
<p><strong>B.</strong> Preparation of the competent cells</p> | <p><strong>B.</strong> Preparation of the competent cells</p> | ||
<p>Reagents:</p> | <p>Reagents:</p> | ||
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<p>Procedure:<br /> | <p>Procedure:<br /> | ||
<strong>Day 1</strong></p> | <strong>Day 1</strong></p> | ||
- | < | + | <p>1. Flame the metal inoculating loop until it is red got and then cools it down.<br /> |
- | + | 2. Scrape off a portion from the top of the frozen glycerol stock [DO NOT THAW].<br /> | |
- | + | 3. Streak it onto the LB plate.<br /> | |
- | + | 4. Put the stock back to -80 oC immediately.<br /> | |
- | + | 5. Leave the plates for 5 min and place them upside down in the 37oC incubator for 16-20 h.</p> | |
- | + | ||
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<p><strong>Day 2</strong></p> | <p><strong>Day 2</strong></p> | ||
- | < | + | <p>6. Pick a single colony into 5 ml of LB medium.<br /> |
- | + | 7. Inoculate the culture overnight at 37oC with shaking at 250 rpm.</p> | |
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<p><strong>Day 3</strong><br /> | <p><strong>Day 3</strong><br /> | ||
8. Inoculate 100 ml LB medium with 1 ml of saturated overnight culture.<br /> | 8. Inoculate 100 ml LB medium with 1 ml of saturated overnight culture.<br /> | ||
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19. Pool all cells into one tube and add 0.5 ml ice-cold 80% glycerol and swirl to mix.<br /> | 19. Pool all cells into one tube and add 0.5 ml ice-cold 80% glycerol and swirl to mix.<br /> | ||
20. Freeze 100 μl aliquots in liquid nitrogen.<br /> | 20. Freeze 100 μl aliquots in liquid nitrogen.<br /> | ||
- | + | 21. Store in -80oC.</p> | |
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<p><strong><a name="a1.6" id="a1.6"></a>1.6 Bacterial Transformation</strong><strong></strong></p> | <p><strong><a name="a1.6" id="a1.6"></a>1.6 Bacterial Transformation</strong><strong></strong></p> | ||
- | < | + | <p>1. Thaw competent cell on ice.<br /> |
- | + | 2. Add 50 - 100 ng DNA to competent cell culture.<br /> | |
- | + | 3. Put in ice for 30 min.<br /> | |
- | + | 4. Heat shock at 42oC for 1 min.<br /> | |
- | + | 5. Put in ice for 2 min.<br /> | |
- | + | 6. Add 1 ml SOC medium.<br /> | |
- | + | 7. Incubate at 37oC for 90 min with shaking (~ 250 rpm).<br /> | |
- | + | 8. Spread plate (with suitable antibiotics)<br /> | |
- | + | 9. Spin down the remaining cells and discard large amount supernatant (1 ml).<br /> | |
- | + | 10. Resuspend the cell pellet and spread plate.<br /> | |
- | + | 11. Incubate at 37oC overnight (preferably ~16 – 20 h).</p> | |
- | + | <p><a href="#top" style="text-decoration : underline; color: #090">Back To Top</a></p> | |
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- | <p><a href="#top">Back To Top</a></p> | + | |
<p><strong><a name="a1.7" id="a1.7"></a>1.7 </strong><strong>Inoculation</strong></p> | <p><strong><a name="a1.7" id="a1.7"></a>1.7 </strong><strong>Inoculation</strong></p> | ||
- | < | + | <p>1. Pick colonies and culture in 2 - 3 ml LB broth with antibiotics.<br /> |
- | + | 2. | |
- | + | Incubate at 37oC for 12 - 16 h with shaking at 250 rpm.</p> | |
- | + | <p><a href="#top" style="text-decoration : underline; color: #090">Back To Top</a></p> | |
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<p><strong><a name="a1.8" id="a1.8"></a>1.8 Plasmid DNA Preparation (Miniprep)</strong></p> | <p><strong><a name="a1.8" id="a1.8"></a>1.8 Plasmid DNA Preparation (Miniprep)</strong></p> | ||
- | < | + | <p>1. Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube.<br /> |
- | + | 2. Add 250 µl Buffer P2 and mix thoroughly by inverting the tube 4 – 6 times and incubate 2 min at room temperature. Do not vortex!<br /> | |
- | + | 3. Add 350 µl Buffer N3 and mix immediately and thoroughly by inverting the tube 4 – 6 times.<br /> | |
- | + | 4. Centrifuge for 10 min at 13,000 rpm in a table-top microcentrifuge. A compact white pellet will form.<br /> | |
- | + | 5. Apply the supernatants from step 4 to the QIAprep spin column<br /> | |
- | + | 6. Centrifuge for 30–60 s. Discard the flow-through.<br /> | |
- | + | 7. Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through.<br /> | |
- | + | 8. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30 – 60 s.<br /> | |
- | + | 9. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.<br /> | |
- | + | 10. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube.<br /> | |
- | + | 11. Add 50 µl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min to elute the DNA.</p> | |
- | + | <p><a href="#top" style="text-decoration : underline; color: #090">Back To Top</a></p> | |
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<p><strong><a name="a1.9" id="a1.9"></a>1.9 Double Digestion</strong></p> | <p><strong><a name="a1.9" id="a1.9"></a>1.9 Double Digestion</strong></p> | ||
+ | <p>1. Mix the components as follows to prepare a 50 μl reaction mixture:</p> | ||
<ul> | <ul> | ||
- | |||
<li>37.6 μl Insert/Vector# + ddH2O*</li> | <li>37.6 μl Insert/Vector# + ddH2O*</li> | ||
- | <li>5 | + | <li>5 μl 10X NEB Buffer3</li> |
- | <li>5 | + | <li>5 μl 10X BSA</li> |
<li>1.2 μl Enzyme 1</li> | <li>1.2 μl Enzyme 1</li> | ||
<li>1.2 μl Enzyme 2</li> | <li>1.2 μl Enzyme 2</li> | ||
</ul> | </ul> | ||
<p># At least 200ng DNA should be added<br /> | <p># At least 200ng DNA should be added<br /> | ||
- | + | * Water is added first and the template the last</p> | |
- | < | + | <p>2. Incubate the reaction mixture at 37oC for 2 h.</p> |
- | + | ||
- | + | ||
- | + | ||
<p>Buffer Chart<br /> | <p>Buffer Chart<br /> | ||
- | + | <a href="http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/buffer_activity_restriction_enzymes.asp">http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/buffer_activity_restriction_enzymes.asp</a></p> | |
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<p><strong><a name="a1.10" id="a1.10"></a>1.10 Gel Extraction*</strong></p> | <p><strong><a name="a1.10" id="a1.10"></a>1.10 Gel Extraction*</strong></p> | ||
- | < | + | <ol> |
<li>Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.</li> | <li>Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.</li> | ||
<li>Weigh the gel slice in a 2 ml centrifuge tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg ~ 100 μl).</li> | <li>Weigh the gel slice in a 2 ml centrifuge tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg ~ 100 μl).</li> | ||
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<li>Place QIAquick column into a clean 1.5 ml microcentrifuge tube.</li> | <li>Place QIAquick column into a clean 1.5 ml microcentrifuge tube.</li> | ||
<li>To elute DNA, add 35 μl of 50oC ddH2O to the center of the QIAquick membrane, wait for 2 min and centrifuge for 2 min at maximum speed.</li> | <li>To elute DNA, add 35 μl of 50oC ddH2O to the center of the QIAquick membrane, wait for 2 min and centrifuge for 2 min at maximum speed.</li> | ||
- | </ | + | </ol> |
<p>*Adopted from CUHK iGEM 2010 protocol.</p> | <p>*Adopted from CUHK iGEM 2010 protocol.</p> | ||
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- | <p><strong><a name="a1.11" id="a1.11"></a>Ligation</strong></p> | + | <p><strong><a name="a1.11" id="a1.11"></a>1.11 Ligation</strong></p> |
+ | <p>3. Mix the components as follows to prepare a 20 μl reaction mixture:</p> | ||
<ul> | <ul> | ||
- | + | <li>2 μl 10X ligation buffer</li> | |
- | <li>2 | + | <li>1 μl T4 DNA ligase</li> |
- | <li>1 | + | |
<li>14 μl Vector</li> | <li>14 μl Vector</li> | ||
- | <li>3 | + | <li>3 μl Insert</li> |
</ul> | </ul> | ||
- | < | + | <p>4. Incubate the reactions at 16oC overnight, or 22oC for 1 h.</p> |
- | + | <p><a href="#top" style="text-decoration : underline; color: #090">Back To Top</a></p> | |
- | + | ||
- | <p><a href="#top">Back To Top</a></p> | + | |
<p><strong><a name="a2.1" id="a2.1"></a>2.1 Cell movement test</strong></p> | <p><strong><a name="a2.1" id="a2.1"></a>2.1 Cell movement test</strong></p> | ||
- | < | + | <ol> |
<li>Pre-culture <em>E. coli</em> for 1 h in LB medium containing 2 μM all-trans retinal.</li> | <li>Pre-culture <em>E. coli</em> for 1 h in LB medium containing 2 μM all-trans retinal.</li> | ||
<li>Transfer 6 μl of pre-cultured solution onto plate with 0.5% agar and 2 μM all-trans retinal</li> | <li>Transfer 6 μl of pre-cultured solution onto plate with 0.5% agar and 2 μM all-trans retinal</li> | ||
<li>Incubate the plate for 24 h under specific wavelength of unidirectional light.</li> | <li>Incubate the plate for 24 h under specific wavelength of unidirectional light.</li> | ||
<li>Measure the cell movement.</li> | <li>Measure the cell movement.</li> | ||
- | </ | + | </ol> |
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<p><strong><a name="a2.2" id="a2.2"></a>2.2 Sensory Rhodopsin-induced Gene Expression Test</strong></p> | <p><strong><a name="a2.2" id="a2.2"></a>2.2 Sensory Rhodopsin-induced Gene Expression Test</strong></p> | ||
- | < | + | <ol> |
<li>Culture <em>E. coli </em>in LB medium with and without light for 24 hrs.</li> | <li>Culture <em>E. coli </em>in LB medium with and without light for 24 hrs.</li> | ||
<li>Measure the OD measurement of the corresponding reporter gene.</li> | <li>Measure the OD measurement of the corresponding reporter gene.</li> | ||
- | </ | + | </ol> |
- | <p><a href="#top">Back To Top</a></p></td> | + | <p><a href="#top" style="text-decoration : underline; color: #090">Back To Top</a></p> |
+ | |||
+ | </td> | ||
</tr> | </tr> | ||
</table> | </table> |
Latest revision as of 16:06, 21 September 2012
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