Team:Hong Kong-CUHK/DOC PROTOCOLS
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+ | <p class="aloveofthunder" style="line-height:normal"><span style="font-size:15px"><strong><a name="top" id="top"></a></strong></span>PROTOCOLS </p> | ||
+ | <p style="font-size:15px; line-height:5px"><strong>1. Cloning</strong></p> | ||
<p><a href="#a1.1">1.1 Gene Amplification with PCR</a></p> | <p><a href="#a1.1">1.1 Gene Amplification with PCR</a></p> | ||
<p><a href="#a1.2">1.2 PCR Purification</a></p> | <p><a href="#a1.2">1.2 PCR Purification</a></p> | ||
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<p><a href="#a1.10">1.10 Gel Extraction</a></p> | <p><a href="#a1.10">1.10 Gel Extraction</a></p> | ||
<p><a href="#a1.11">1.11 Ligation</a></p> | <p><a href="#a1.11">1.11 Ligation</a></p> | ||
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<p style="font-size:15px"><strong>2. Functional test</strong></p> | <p style="font-size:15px"><strong>2. Functional test</strong></p> | ||
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<p><a href="#a2.1">2.1 Cell Movement Test</a></p> | <p><a href="#a2.1">2.1 Cell Movement Test</a></p> | ||
<p><a href="#a2.2">2.2 Sensory Rhodopsin-induced Gene Expression Test</a></p> | <p><a href="#a2.2">2.2 Sensory Rhodopsin-induced Gene Expression Test</a></p> | ||
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<hr /> | <hr /> | ||
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<p> </p> | <p> </p> | ||
<p><strong><a name="a1.1" id="a1.1"></a><span class="GREEN">1.1 Gene Amplification with PCR</span></strong></p> | <p><strong><a name="a1.1" id="a1.1"></a><span class="GREEN">1.1 Gene Amplification with PCR</span></strong></p> | ||
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<p>1. Put the reaction tubes on ice.<br /> | <p>1. Put the reaction tubes on ice.<br /> | ||
2. Add the following components into each reaction tube:</p> | 2. Add the following components into each reaction tube:</p> | ||
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4°C</p> | 4°C</p> | ||
<p><a href="#top" style="text-decoration : underline; color: #090">Back To Top</a></p> | <p><a href="#top" style="text-decoration : underline; color: #090">Back To Top</a></p> | ||
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<p> </p> | <p> </p> | ||
<p><strong><a name="a1.2" id="a1.2"></a><span class="GREEN">1.2 PCR Purification*</span></strong></p> | <p><strong><a name="a1.2" id="a1.2"></a><span class="GREEN">1.2 PCR Purification*</span></strong></p> | ||
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<ol> | <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> | ||
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<p>*Protocol adopted from QIAquick PCR purification kit protocol</p> | <p>*Protocol adopted from QIAquick PCR purification kit protocol</p> | ||
<p><a href="#top" style="text-decoration : underline; color: #090">Back To Top</a></p> | <p><a href="#top" style="text-decoration : underline; color: #090">Back To Top</a></p> | ||
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<p> </p> | <p> </p> | ||
<p class="GREEN"><strong><a name="a1.3" id="a1.3"></a>1.3 Overlapping PCR</strong></p> | <p class="GREEN"><strong><a name="a1.3" id="a1.3"></a>1.3 Overlapping PCR</strong></p> | ||
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<p>1. Put the reaction tubes on ice.<br /> | <p>1. Put the reaction tubes on ice.<br /> | ||
2. Add the following components into each reaction tube:</p> | 2. Add the following components into each reaction tube:</p> | ||
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4°C</p> | 4°C</p> | ||
<p><a href="#top" style="text-decoration : underline; color: #090">Back To Top</a></p> | <p><a href="#top" style="text-decoration : underline; color: #090">Back To Top</a></p> | ||
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<p> </p> | <p> </p> | ||
<p><strong><a name="a1.4" id="a1.4"></a><span class="GREEN">1.4 Agarose Gel Electrophoresis</span></strong></p> | <p><strong><a name="a1.4" id="a1.4"></a><span class="GREEN">1.4 Agarose Gel Electrophoresis</span></strong></p> | ||
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<p><strong>A. </strong>Cast gel</p> | <p><strong>A. </strong>Cast gel</p> | ||
<p>1. Dissolve 0.55 g agarose into 55 ml 0.5X TBE buffer.<br /> | <p>1. Dissolve 0.55 g agarose into 55 ml 0.5X TBE buffer.<br /> | ||
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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> | 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><a href="#top" style="text-decoration : underline; color: #090">Back To Top</a></p> | ||
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<p> </p> | <p> </p> | ||
<p><strong><a name="a1.5" id="a1.5"></a><span class="GREEN">1.5 Preparation of Competent Cells</span></strong></p> | <p><strong><a name="a1.5" id="a1.5"></a><span class="GREEN">1.5 Preparation of Competent Cells</span></strong></p> | ||
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<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 /> | <p>1. Autoclave glassware filled with 3/4 dd-H2O to remove most residual detergent.<br /> | ||
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12. Transfer the culture into two pre-chilled 50ml falcon.<br /> | 12. Transfer the culture into two pre-chilled 50ml falcon.<br /> | ||
13. Centrifuge at 2700 x g for 10 min at 4oC<br /> | 13. Centrifuge at 2700 x g for 10 min at 4oC<br /> | ||
- | 14. Remove the medium, resuspend the cell pellet with 1.6 ml ice-cold 100 mM CaCl2 by swirling on ice gently.<br /> | + | 14. Remove the medium, and resuspend the cell pellet with 1.6 ml ice-cold 100 mM CaCl2 by swirling on ice gently.<br /> |
15. Incubate on ice for 30 min.<br /> | 15. Incubate on ice for 30 min.<br /> | ||
16. Centrifuge at 2700 x g for 10 minutes at 4oC.<br /> | 16. Centrifuge at 2700 x g for 10 minutes at 4oC.<br /> | ||
- | 17. Remove the medium, resuspend the cell pellet with 1.6 ml ice-cold 100 mM CaCl2 by swirling on ice gently.<br /> | + | 17. Remove the medium, and resuspend the cell pellet with 1.6 ml ice-cold 100 mM CaCl2 by swirling on ice gently.<br /> |
18. Incubate on ice for 20 min.<br /> | 18. Incubate on ice for 20 min.<br /> | ||
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 /> | ||
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21. Store in -80oC.</p> | 21. Store in -80oC.</p> | ||
<p><a href="#top" style="text-decoration : underline; color: #090">Back To Top</a></p> | <p><a href="#top" style="text-decoration : underline; color: #090">Back To Top</a></p> | ||
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<p> </p> | <p> </p> | ||
<p><strong><a name="a1.6" id="a1.6"></a><span class="GREEN">1.6 Bacterial Transformation</span></strong></p> | <p><strong><a name="a1.6" id="a1.6"></a><span class="GREEN">1.6 Bacterial Transformation</span></strong></p> | ||
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<p>1. Thaw competent cell on ice.<br /> | <p>1. Thaw competent cell on ice.<br /> | ||
2. Add 50 - 100 ng DNA to competent cell culture.<br /> | 2. Add 50 - 100 ng DNA to competent cell culture.<br /> | ||
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11. Incubate at 37oC overnight (preferably ~16 – 20 h).</p> | 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> | <p><a href="#top" style="text-decoration : underline; color: #090">Back To Top</a></p> | ||
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<p> </p> | <p> </p> | ||
<p><strong><a name="a1.7" id="a1.7"></a><span class="GREEN">1.7 </span></strong><span class="GREEN"><strong>Inoculation</strong></span></p> | <p><strong><a name="a1.7" id="a1.7"></a><span class="GREEN">1.7 </span></strong><span class="GREEN"><strong>Inoculation</strong></span></p> | ||
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<p>1. Pick colonies and culture in 2 - 3 ml LB broth with antibiotics.<br /> | <p>1. Pick colonies and culture in 2 - 3 ml LB broth with antibiotics.<br /> | ||
2. | 2. | ||
Incubate at 37oC for 12 - 16 h with shaking at 250 rpm.</p> | 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> | <p><a href="#top" style="text-decoration : underline; color: #090">Back To Top</a></p> | ||
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<p> </p> | <p> </p> | ||
<p><strong><a name="a1.8" id="a1.8"></a><span class="GREEN">1.8 Plasmid DNA Preparation (Miniprep)</span></strong></p> | <p><strong><a name="a1.8" id="a1.8"></a><span class="GREEN">1.8 Plasmid DNA Preparation (Miniprep)</span></strong></p> | ||
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<p>1. Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube.<br /> | <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 /> | 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 /> | ||
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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> | 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> | <p><a href="#top" style="text-decoration : underline; color: #090">Back To Top</a></p> | ||
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<p> </p> | <p> </p> | ||
<p><strong><a name="a1.9" id="a1.9"></a><span class="GREEN">1.9 Double Digestion</span></strong></p> | <p><strong><a name="a1.9" id="a1.9"></a><span class="GREEN">1.9 Double Digestion</span></strong></p> | ||
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<p>1. Mix the components as follows to prepare a 50 μl reaction mixture:</p> | <p>1. Mix the components as follows to prepare a 50 μl reaction mixture:</p> | ||
<ul> | <ul> | ||
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<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> | <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> | ||
<p><a href="#top" style="text-decoration : underline; color: #090">Back To Top</a></p> | <p><a href="#top" style="text-decoration : underline; color: #090">Back To Top</a></p> | ||
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<p> </p> | <p> </p> | ||
<p><strong><a name="a1.10" id="a1.10"></a><span class="GREEN">1.10 Gel Extraction*</span></strong></p> | <p><strong><a name="a1.10" id="a1.10"></a><span class="GREEN">1.10 Gel Extraction*</span></strong></p> | ||
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<ol> | <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> | ||
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<p> </p> | <p> </p> | ||
<p><strong><a name="a1.11" id="a1.11"></a><span class="GREEN">1.11 Ligation</span></strong></p> | <p><strong><a name="a1.11" id="a1.11"></a><span class="GREEN">1.11 Ligation</span></strong></p> | ||
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<p>3. Mix the components as follows to prepare a 20 μl reaction mixture:</p> | <p>3. Mix the components as follows to prepare a 20 μl reaction mixture:</p> | ||
<ul> | <ul> | ||
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<p><a href="#top" style="text-decoration : underline; color: #090">Back To Top</a></p> | <p><a href="#top" style="text-decoration : underline; color: #090">Back To Top</a></p> | ||
<p> </p> | <p> </p> | ||
- | + | <p class="GREEN"><strong><a name="a2.1" id="a2.1"></a>2.1 Cell movement test</strong></p> | |
- | <p class="GREEN"><strong><a name="a2.1" id="a2.1"></a>2.1 Cell movement test</strong> | + | |
<ol> | <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> | ||
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<p><a href="#top" style="text-decoration : underline; color: #090">Back To Top</a></p> | <p><a href="#top" style="text-decoration : underline; color: #090">Back To Top</a></p> | ||
<p> </p> | <p> </p> | ||
- | + | <p class="GREEN"><strong><a name="a2.2" id="a2.2"></a>2.2 Sensory Rhodopsin-induced Gene Expression Test</strong></p> | |
- | <p class="GREEN"><strong><a name="a2.2" id="a2.2"></a>2.2 Sensory Rhodopsin-induced Gene Expression Test</strong> | + | |
<ol> | <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> |
Latest revision as of 02:25, 27 September 2012
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1. Cloning 1.1 Gene Amplification with PCR 1.4 Agarose Gel Electrophoresis 1.5 Preparation of Competent Cells 1.8 Plasmid DNA Extraction (Miniprep) 2. Functional test 2.2 Sensory Rhodopsin-induced Gene Expression Test
1.1 Gene Amplification with PCR 1. Put the reaction tubes on ice.
3. Mix well and run the following program:
2-step PCR Cycling Program 25–35 cycles Final extension: Hold:
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. *Protocol adopted from QIAquick PCR purification kit protocol
1. Put the reaction tubes on ice.
3. Mix well and run the following program: 2-step PCR Cycling Program 25–35 cycles: Hold:
1.4 Agarose Gel Electrophoresis A. Cast gel 1. Dissolve 0.55 g agarose into 55 ml 0.5X TBE buffer. B. Run gel 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.
1.5 Preparation of Competent Cells A. Preparation of detergent-free glassware and media 1. Autoclave glassware filled with 3/4 dd-H2O to remove most residual detergent. B. Preparation of the competent cells Reagents:
Procedure: 1. Flame the metal inoculating loop until it is red got and then cools it down. Day 2 6. Pick a single colony into 5 ml of LB medium. Day 3
1. Thaw competent cell on ice.
1. Pick colonies and culture in 2 - 3 ml LB broth with antibiotics.
1.8 Plasmid DNA Preparation (Miniprep) 1. Resuspend pelleted bacterial cells in 250 µl Buffer P1 and transfer to a microcentrifuge tube.
1. Mix the components as follows to prepare a 50 μl reaction mixture:
# At least 200ng DNA should be added 2. Incubate the reaction mixture at 37oC for 2 h. Buffer Chart
*Adopted from CUHK iGEM 2010 protocol.
3. Mix the components as follows to prepare a 20 μl reaction mixture:
4. Incubate the reactions at 16oC overnight, or 22oC for 1 h.
2.2 Sensory Rhodopsin-induced Gene Expression Test
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