Difference between revisions of "Team:SiCAU-China/Experiments"

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<div class="title"><img src="https://static.igem.org/mediawiki/2017/2/23/T-SICAU-protocol_title.jpg" /></div>
 
<div class="title"><img src="https://static.igem.org/mediawiki/2017/2/23/T-SICAU-protocol_title.jpg" /></div>
 
<div style="clear:both;height:60px;"></div>
 
<div style="clear:both;height:60px;"></div>
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<section id="mcc"><h1><img src="https://static.igem.org/mediawiki/2017/6/6f/T-SICAU-Fire_paint1.jpg" /> Making Competent Cells</h1>
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<section id="mcc"><div class="p-size">&nbsp;</div>
 +
<h1><img src="https://static.igem.org/mediawiki/2017/6/6f/T-SICAU-Fire_paint1.jpg" /> Making Competent Cells</h1>
 
<table>
 
<table>
 
<tr><th align="center">Step</th><th>Method</th></tr>
 
<tr><th align="center">Step</th><th>Method</th></tr>
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</section>
 
</section>
 
<div style="clear:both;height:30px;"></div>
 
<div style="clear:both;height:30px;"></div>
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<section id="pcr"><h1><img src="https://static.igem.org/mediawiki/2017/2/28/T-SICAU-fire_paint2.jpg" /> PCR</h1>
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<section id="pcr"><div class="p-size">&nbsp;</div>
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<h1><img src="https://static.igem.org/mediawiki/2017/2/28/T-SICAU-fire_paint2.jpg" /> PCR</h1>
 
<div class="p-size">1. Mix the following:
 
<div class="p-size">1. Mix the following:
 
<div style="clear"></div>
 
<div style="clear"></div>
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3. Agarose Gel Electrophoresis for confirmation.
 
3. Agarose Gel Electrophoresis for confirmation.
 
</div></section>
 
</div></section>
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<section id="min"><h1><img src="https://static.igem.org/mediawiki/2017/2/20/T-SICAU-fire_paint3.jpg" />  Miniprep</h1>
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<section id="min"><div class="p-size">&nbsp;</div>
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<h1><img src="https://static.igem.org/mediawiki/2017/2/20/T-SICAU-fire_paint3.jpg" />  Miniprep</h1>
 
<table>
 
<table>
 
<tr><th align="center">Step</th><th>Method</th></tr>
 
<tr><th align="center">Step</th><th>Method</th></tr>
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</section>
 
</section>
 
<div style="clear:both;height:30px;"></div>
 
<div style="clear:both;height:30px;"></div>
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<section id="ep"><h1><img src="https://static.igem.org/mediawiki/2017/d/d8/T-SICAU-fire_paint4.jpg" /> Electrophoresis</h1>
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<section id="ep"><div class="p-size">&nbsp;</div>
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<h1><img src="https://static.igem.org/mediawiki/2017/d/d8/T-SICAU-fire_paint4.jpg" /> Electrophoresis</h1>
 
<table>
 
<table>
 
<tr><td align="center">1. Agalose Gel casting</td><td >
 
<tr><td align="center">1. Agalose Gel casting</td><td >
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</section>
 
</section>
 
<div style="clear:both;height:30px;"></div>
 
<div style="clear:both;height:30px;"></div>
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<section id="red"><h1><img src="https://static.igem.org/mediawiki/2017/1/1d/T-SICAU-fire_paint5.jpg" /> Restriction Enzyme Digestion</h1>
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<section id="red"><div class="p-size">&nbsp;</div>
 +
<h1><img src="https://static.igem.org/mediawiki/2017/1/1d/T-SICAU-fire_paint5.jpg" /> Restriction Enzyme Digestion</h1>
 
<div class="p-size">
 
<div class="p-size">
 
1. Mix the following:<br/>
 
1. Mix the following:<br/>
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2. Let stand for 1 hour at 37 °C.<br/>
 
2. Let stand for 1 hour at 37 °C.<br/>
 
</div></section>
 
</div></section>
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<section id="ligation"><h1><img src="https://static.igem.org/mediawiki/2017/f/f7/T-SICAU-fire_paint6.jpg" /> Ligation</h1>
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<section id="ligation"><div class="p-size">&nbsp;</div>
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<h1><img src="https://static.igem.org/mediawiki/2017/f/f7/T-SICAU-fire_paint6.jpg" /> Ligation</h1>
 
<table>
 
<table>
 
<tr><td align="center">1</td><td>Mix the vector DNA and the insert DNA (the vector and the insert at 1 : 5-10).</td></tr>
 
<tr><td align="center">1</td><td>Mix the vector DNA and the insert DNA (the vector and the insert at 1 : 5-10).</td></tr>
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</table>
 
</table>
 
</section>
 
</section>
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<section id="transformation"><h1><img src="https://static.igem.org/mediawiki/2017/e/e4/T-SICAU-fire_paint7.jpg" />Transformation</h1>
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<section id="transformation"><div class="p-size">&nbsp;</div>
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<h1><img src="https://static.igem.org/mediawiki/2017/e/e4/T-SICAU-fire_paint7.jpg" />Transformation</h1>
 
<table>
 
<table>
 
<tr><td align="center">1</td><td>Add 2ul DNA to 150ul chemically conpetent cells on ice (set negative control by using chemically competent E.coli cells without plasmids).</td></tr>
 
<tr><td align="center">1</td><td>Add 2ul DNA to 150ul chemically conpetent cells on ice (set negative control by using chemically competent E.coli cells without plasmids).</td></tr>
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</section>
 
</section>
 
<div style="clear:both;height:30px;"></div>
 
<div style="clear:both;height:30px;"></div>
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<section id="fm"><h1><img src="https://static.igem.org/mediawiki/2017/d/df/T-SICAU-fire_paint8.jpg" /> Fluorometric measurement</h1>
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<section id="fm"><div class="p-size">&nbsp;</div>
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<h1><img src="https://static.igem.org/mediawiki/2017/d/df/T-SICAU-fire_paint8.jpg" /> Fluorometric measurement</h1>
 
<div class="p-size">1. Add 2ml of the sample to quartz cell.<br/>
 
<div class="p-size">1. Add 2ml of the sample to quartz cell.<br/>
 
<div style="clear:both;height:30px;"></div>
 
<div style="clear:both;height:30px;"></div>
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3. Measure by Fluorospectro photometer model: F-4600 FL Spectrophotometer<br/>
 
3. Measure by Fluorospectro photometer model: F-4600 FL Spectrophotometer<br/>
 
</div></section>
 
</div></section>
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<section id="hplc"><h1><img src="https://static.igem.org/mediawiki/2017/3/38/T-SICAU-fire_paint9.jpg" /> HPLC（High Performance Liquid Chromatograpy） </h1>
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<section id="hplc"><div class="p-size">&nbsp;</div>
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<h1><img src="https://static.igem.org/mediawiki/2017/3/38/T-SICAU-fire_paint9.jpg" /> HPLC（High Performance Liquid Chromatograpy） </h1>
 
<div class="p-size">
 
<div class="p-size">
 
Inject 10μL of the sample into column.HPLC condition will show below：<br/>
 
Inject 10μL of the sample into column.HPLC condition will show below：<br/>

Revision as of 16:24, 1 November 2017

top

PCR

Electrophoresis

Restriction Enzyme
Digestion

Ligation

Transformation

Fluorometric measurement

HPLC

 

Making Competent Cells

StepMethod
1Streak E.coli cells on an LB plate.
2Allow cells to grow at 37°C overnight.
3Place one colony in 10 mL LB media(+antibiotic selection if necessary), grow overnight at 37°C.
4Take 2 ml LB media and save for blank. Transfer 500 µL overnight culture into 50 mL LB media in 250 mL flask.
5Allow cell to grow at 37°C (200 rpm), until OD600= 0.5 (~2-3 hours).
6Place cells on ice for 30 minutes.
7Transfer cells to a centrifuge tube (50 mL), and centrifuge cells at 4°C for 10 minutes at 4,000×g.
8Pour off media and resuspend cells in 12 mL of cold TB.
9Centrifuge cells at 4°C for 10 minutes at 4,000×g.
10Pour supernatant and resuspend cells (by pipetting) in 4 mL of cold TB and 280 µL of DMSO. Transfer 100 µL to 1.5 mL tube.
11 Ependorff tubes placed on ice. Freeze the cells in liquid nitrogen. Cells stored at -80°C can be used for transformation for up to ~6 months.
 

PCR

1. Mix the following:
Name Volume
PrimeSTAR® Max DNA Polymerase 10µL
Template DNA 1µL
Primer Forward (10 µM) 1 µL
Primer Reverse (10 µM) 1µL
ddH2O up to 20µL
Total 20 µL
2. Put samples into Thermal Cyclers and run the following steps:
PreDenature Denature Anealing Extension cycle
98 °C 98 °C Tm-5 °C 72 °C --
2 min 30 sec 30 sec 15 sec /kb 30 cycles
3. Agarose Gel Electrophoresis for confirmation.
 

Miniprep

StepMethod
1Pellet 1-4ml of overnight culture by centrifugation at 12,000×g for 1 minute. Discard the supertanant completely.
2Add 250ul of SolutionⅠto the pellet to resuspend bacteria cells.
3Add 250ul of SolutionⅡ, mix gently by inverting the tube 7-8 times until the solution becomes clear. The time should be no longer than 5 minutes.
4Add 350ul of SolutionⅢ, mix gently by inverting the tube 7-8 times.
5Centrifuge at 12,000rpm for 10 minutes.
6Place spin column into a 1.5ml collection tube. Transfer supernatant in the step above to the column. Centrifuge at 12,000rpm for 1 minute. Discard the filtrate from the 2ml microfuge tube.
7Return the column to the 2ml microfuge tube and add 600ul of Buffer PW. Centrifuge at 12,000×g for 1 minute. Discard the filtrate from the 2ml microfuge tube.
8Return the column to the 2ml microfuge tube and add 600ul of Buffer PW. Centrifuge at 12,000×g for 1 minute. Discard the filtrate from the 2ml microfuge tube.
9Place the column back into the 2ml microfuge tube. Centrifuge at 12,000×g for 2 minute. Discard the filtrate and the 2ml microfuge tube.
10Place the spin column onto dry block heater for 8 minute.
11Transfer the column into a clean 1.5ml microfuge tube .Add 60-80ul of Eluent or deionized water to the center of the membrane to elute the DNA. Let it stand for 1 minute at room temperature. Centrifuge at 12,000×g for 1 minute. Repeat this operation two times. Note: Pre-warm the Eluent or deionized water at 50℃ will generally improve elution efficiency.
 

Electrophoresis

1. Agalose Gel casting 1) Measure out the appropriate mass of agarose into glass bottle with the appropriate volume of TAE buffer.
2) Microwave until the agarose is fully melted.
3) Pour the agarose solution into the gelbox and let it cool for about 30 minutes, until the gel is solid.
4) Remove comb.
2.Running agalose gel 1) Load 5 μL prepared 1kbp ladder.
2) Mix DNA solution with loading buffer (5x).
3) Load it into agalose gel.
4) Run the gel at ~110 volts for 30 minutes.
3.Visualizing agarose gels
 1) Remove gel from gel box.
2) Soak the gel in ethidium bromide solution.
3) Place the gel in Trans-Illuminator and turn on UV light after make sure the door closing.
4) Print the picture.
5) Remove gel and throw in trash.
 

Restriction Enzyme Digestion

1. Mix the following:
Name Volume
Sample DNA 4 µg
Restriction Enzyme
(EcoRI, XbaI, SpeI, PstI)		 0.5µL
10x Buffer 1 µL
ddH2O up to 10 µL
total 10 µL
2. Let stand for 1 hour at 37 °C.
 

Ligation

1Mix the vector DNA and the insert DNA (the vector and the insert at 1 : 5-10).
2Add 1ul Exnase® II Ligase and 2ul CE II Buffer(5x).
3Incubate at 16 °C for 1 hour (or at 4 °C for overnight).
 

Transformation

1Add 2ul DNA to 150ul chemically conpetent cells on ice (set negative control by using chemically competent E.coli cells without plasmids).
2Incubate on ice for 30 minutes.
3Heat shock at 42℃ for exactly 90 seconds.
4Place samples back on ice for 2 minutes.
5Operating in the clean bench, add 900ul of LB broth per tube.
6Incubate at 37℃ for 60 minutes, shaking.
7Centrifuge at 4000rpm for 1 minute.
8Operating in the clean bench, discard the supertanant (about 700ul) and resuspend bacteria cells.
9Use the inoculating loop to load bacteria liquid then streak on the LB plate (+antibiotic selection if necessary).
10Place plates upside down and incubate at 37℃ overnight.
 

Fluorometric measurement

1. Add 2ml of the sample to quartz cell.
2. Setting measurement parameter:
490nm 450nm 550nm 1200nm/min 2.5nm 2.5nm 900V
EX wavelength
EM start wavelength
EM end wavelength
Scan speed
EX slit
EM slit
PMT voltage
3. Measure by Fluorospectro photometer model: F-4600 FL Spectrophotometer
 

HPLC（High Performance Liquid Chromatograpy）

Inject 10μL of the sample into column.HPLC condition will show below：
HPLCAgilent 1290 Infinity LC
ColumnZORBAX Eclipse® XDB-C18
Total Flow1mL/min
Pump APump B=H2O
temperature25℃
Pump A:Pump B=H2O:MeOH=0.6:0.4

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