Team:LZU-China/Notebook
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+ | /***** notebook ****/ | ||
+ | table{background:none;} | ||
+ | table td{color:#FFF; font-size:14px; padding-left:3px;} | ||
+ | h1,h2,h3,h4,h5,h6{color:#FFF;} | ||
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<a href="https://2013.igem.org/Team:LZU-China/Project"><li>Project</li></a> | <a href="https://2013.igem.org/Team:LZU-China/Project"><li>Project</li></a> | ||
<a href="https://2013.igem.org/Team:LZU-China/Parts"><li>Parts</li></a> | <a href="https://2013.igem.org/Team:LZU-China/Parts"><li>Parts</li></a> | ||
- | <a href="https://2013.igem.org/Team:LZU-China/Notebook"><li class="selected"> | + | <a href="https://2013.igem.org/Team:LZU-China/Notebook"><li class="selected">Notebook</li></a> |
<a href="https://2013.igem.org/Team:LZU-China/Safety"><li>Safety</li></a> | <a href="https://2013.igem.org/Team:LZU-China/Safety"><li>Safety</li></a> | ||
<a href="https://2013.igem.org/Team:LZU-China/Attributions"><li>Attributions</li></a> | <a href="https://2013.igem.org/Team:LZU-China/Attributions"><li>Attributions</li></a> | ||
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<div class="main-center" id="main-center"> | <div class="main-center" id="main-center"> | ||
<div class="center-left" id="center-left"> | <div class="center-left" id="center-left"> | ||
- | <h1> | + | <a href="#h-events"><h1>Events</h1></a> |
- | <h1> | + | <a href="#h-protocol"><h1>protocol</h1></a> |
- | + | <a href="#h-bc"><h2>Bacteria Culture</h2></a> | |
+ | <a href="#h-vp"><h2>Violacein Protocols</h2></a> | ||
+ | </div> | ||
<div class="center-right"> | <div class="center-right"> | ||
- | < | + | |
- | + | <h2> <span class="mw-headline" id="h-events">Events</span></h2> | |
- | + | <h3> <span class="mw-headline" id="July_1-14">July 1-14</span></h3> | |
- | + | <p>Amplified IκB fragment from the Plasmid of by Polymerase Chain Reaction(PCR). | |
- | + | </p> | |
- | + | <h3> <span class="mw-headline" id="July_15-25">July 15-25</span></h3> | |
- | + | <p>Amplified GFP fragment from the Plasmid of pEGFP-N1 by Polymerase Chain Reaction(PCR). | |
- | + | </p> | |
- | + | <h3> <span class="mw-headline" id="July_25-August_1">July 25-August 1</span></h3> | |
- | + | <p>BIT Team's member Zhou Xiaoyu visited our lab. We designed and constructed IκB-GFP fusion fragments together. | |
+ | </p> | ||
+ | <h3> <span class="mw-headline" id="August_1-15">August 1-15 iGEM Lab Opening Day</span></h3> | ||
+ | <p>Used Overlap PCR techniques to build IκB-GFP fusion fragment. Meanwhile, our team held a event which named the Opening Day of iGEM Lab for two weeks. | ||
+ | </p> | ||
+ | <h3> <span class="mw-headline" id="August_3-4">August 3-4</span></h3> | ||
+ | <p>LZU-China Team member Li Xiaomeng was invited to interchange with the Peking iGEM 2013 Team. | ||
+ | </p> | ||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/2013/0/0c/IMG_6928.jpg"/> | ||
+ | |||
+ | <h3> <span class="mw-headline" id="August_4-11_Interchange_and_Helping_BIT">August 4-11 Interchange and Helping BIT </span></h3> | ||
+ | <p>LZU-China Team member Li Xiaomeng was invited to interchange with the BIT iGEM 2013 Team. And help them to characterize and construct a part of Cr sensor. | ||
+ | </p> | ||
+ | |||
+ | |||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/2013/1/18/IMG_0102.jpg"/> | ||
+ | <img src="https://static.igem.org/mediawiki/2013/1/1e/IMG_6942.jpg"/><br><br> | ||
+ | |||
+ | <img src="https://static.igem.org/mediawiki/2013/7/71/IMG_6943.jpg"/> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <h3> <span class="mw-headline" id="August_16-30">August 16-30</span></h3> | ||
+ | <p>The primers synthesized two complementary single stranded DNA with NF-κB binding element. And then combined a completely DNA fragment. | ||
+ | Used restriction enzymolysis, connection, and transformation to build NF-κB-IκB-GFP fragment. | ||
+ | </p> | ||
+ | <h3> <span class="mw-headline" id="September_1-16">September 1-16</span></h3> | ||
+ | <p>Put our part into the backbone with the BioBrick prefix and suffix. And sequenced the Plasmid which will be shipped to iGEM. | ||
+ | </p><p><br /> | ||
+ | </p> | ||
+ | <h2> <span class="mw-headline" id="h-protocol">Molecular Cloning Protocols</span></h2> | ||
+ | <h3> <span class="mw-headline" id="Digestion">Digestion</span></h3> | ||
+ | <h4> <span class="mw-headline" id="Material">Material</span></h4> | ||
+ | <ul><li>EcoRI, BglII, PstI, XhoI, SdaI,XbaI,10X Tango buffer,10X O buffer, DNA,sterile water | ||
+ | </li></ul> | ||
+ | <h4> <span class="mw-headline" id="Digestion_temperature">Digestion temperature</span></h4> | ||
+ | <ul><li>37℃ | ||
+ | </li></ul> | ||
+ | <h4> <span class="mw-headline" id="Time_period">Time period</span></h4> | ||
+ | <ol><li>Plasmid: 12-16h(the higher the concentration, the longer time it takes. | ||
+ | </li><li>DNA fragment:12-16h(the higher the concentration, the longer time it takes.Since it is more difficult to digest DNA than plasmid, digesting DNA requires more time. | ||
+ | </li></ol> | ||
+ | <h4> <span class="mw-headline" id="System_amount">System amount</span></h4> | ||
+ | <ol><li>50ul system is preferred if there is subsequent operations. If digestion is only for identification, 20ul system is used. | ||
+ | </li><li>During the double digestion, the optimum buffer system which is suitable for both enzymes should be utilized. Without such shared system, digestion of two different enzymes is usually separated. | ||
+ | </li></ol> | ||
+ | <h4> <span class="mw-headline" id="System">System</span></h4> | ||
+ | <dl><dd><dl><dd><dl><dd><table border="1"> | ||
+ | <tr> | ||
+ | <td width="200">Enzyme | ||
+ | </td><td width="150">1μl | ||
+ | </td></tr> | ||
+ | <tr> | ||
+ | <td>10X Tango buffer or 10X O buffer</td><td>2μl | ||
+ | </td></tr> | ||
+ | <tr> | ||
+ | <td>DNA </td><td>≤1μg | ||
+ | </td></tr> | ||
+ | <tr> | ||
+ | <td>Sterile water</td><td>up to 20μl | ||
+ | </td></tr></table></dd></dl></dd></dl></dd></dl> | ||
+ | <h3> <span class="mw-headline" id="Gel_extraction">Gel extraction</span></h3> | ||
+ | <h4> <span class="mw-headline" id="Material_2">Material</span></h4> | ||
+ | <ul><li>Agarose Gel DNA Extraction Kit (centrifugation) | ||
+ | </li></ul> | ||
+ | <h4> <span class="mw-headline" id="Experimental_Procedure">Experimental Procedure</span></h4> | ||
+ | <ol><li>Add 500uL balance liquid(BL) to the spin column(CA2). Centrifuge at 12,000 x g for 1 min at room temperature. Discard the flow-through and put the column back to the collection tube.(optional) | ||
+ | </li><li>Excise the DNA fragment from the agarose gel and weigh it in a clean centrifuge tube (A gel slice of 100 mg approximately equals to 100 µL). | ||
+ | </li><li>Add 3 volume of sol solution(PN) to the centrifuge tube and incubate the mixture at 50℃ for 10 min with mixing the tube by tapping the bottom of the tube occationally till the gel has melted completely. Cool the tube to RT. | ||
+ | </li><li>Transfer the mixture to an spin column (CA2) within a collection tube. Centrifuge at 12,000 x g for 1 min at room temperature. Discard the flow-through and put the column back to the collection tube. | ||
+ | </li><li>Add 600 µL DNA Wash Buffer (PW) to the column and centrifuge at 12,000 x g for 1 min at room temperature. Discard the flow through and put the column back to the collection tube. | ||
+ | </li><li>Repeat step 5. | ||
+ | </li><li>Put the column back to the collection tube. Centrifuge at 12,000 x g for 2 min at room temperature to discard the PW totally. Keep the lid open for several minutes. | ||
+ | </li><li>Put the column back to the clean collection tube. Add ddH2O to the column vacantly and keep it at room temperature for 2 minutes. Centrifuge at 12,000 x g for 2 min at room temperature to collect the DNA solution. | ||
+ | </li><li>Transfer the eluted solution to CA2 and keep it at room temperature for 2minutes. Centrifuge at 12,000 x g for 2 min at room temperature. | ||
+ | </li><li>Gel electrophoresis to verify the correctness and concentration of bands. | ||
+ | </li></ol> | ||
+ | <h4> <span class="mw-headline" id="Important_Notes">Important Notes</span></h4> | ||
+ | <ol><li>If the balance liquid turns turbid, incubate it at 37℃ for several minutes. | ||
+ | </li><li>When cutting the gel, keep the UV irradiation time period as short as possible, otherwise DNA would be damaged. | ||
+ | </li><li>For those DNA fragment shorter than 100bp or larger than 10kb, the sol solution volume should be increased as well as the time period for adsorption and elution. | ||
+ | </li><li>Recovery depends on the initial DNA amount and elution volume. The less the DNA, the less the elution volume, the smaller of recovery. | ||
+ | </li></ol> | ||
+ | <h3> <span class="mw-headline" id="PCR">PCR</span></h3> | ||
+ | <h4> <span class="mw-headline" id="Reagents">Reagents</span></h4> | ||
+ | <ul><li>Pfu enzymes | ||
+ | </li><li>Tap enzymes | ||
+ | </li></ul> | ||
+ | <h4> <span class="mw-headline" id="Experiment_system">Experiment system</span></h4> | ||
+ | <ul><li>Experiment system with Pfu enzymes (Mostly 50uL) | ||
+ | </li></ul> | ||
+ | <ul><li>Experiment system with taq enzymes: | ||
+ | </li></ul> | ||
+ | <ul><li>Reaction temperature: | ||
+ | </li></ul> | ||
+ | <pre> Two-step method: | ||
+ | Predenature | ||
+ | 94℃,2min | ||
+ | Denature and extension | ||
+ | { | ||
+ | 94℃ 30sec | ||
+ | 72℃ 60sec/kb | ||
+ | } | ||
+ | × 25-45 cycles | ||
+ | 4℃ | ||
+ | </pre> | ||
+ | <hr /> | ||
+ | <pre> Three-step method: | ||
+ | Predenature | ||
+ | 94℃,2min | ||
+ | Denature,annealing and extension | ||
+ | 94℃ 30sec | ||
+ | { | ||
+ | (Tm-5)℃ 30sec | ||
+ | 72℃ 60sec/kb | ||
+ | } | ||
+ | × 25-45 cycles | ||
+ | 4℃ | ||
+ | </pre> | ||
+ | <h4> <span class="mw-headline" id="Important_Notes_2">Important Notes</span></h4> | ||
+ | <ol><li>Pfu enzymes should be operated in ice. | ||
+ | </li><li>Two-step method is preferred if primer Tm is relatively high. | ||
+ | </li><li>1.2% agarose gel is used to separate 1kb-5kb DNA; 0.8% agarose gel is used to separate 3k-10k DNA; 2.5-3% agarose gel is used to separate DNA longer than 100bp. | ||
+ | </li><li>PCR reaction system varies from 20uL to 100uL. Under most cases, 20uL system is preferred as long as PCR is for identification only. However, when subsequent experiments such as tailing, digestion which require certain amount of DNA are necessary, 50uL system is usually preferred. | ||
+ | </li><li>Pfu enzymes are preferred if the purpose of PCR is for gene amplification. At the same time, PCR for identification or tailing usually requires taq enzymes. | ||
+ | </li></ol> | ||
+ | <h3> <span class="mw-headline" id="PCR_Purification">PCR Purification</span></h3> | ||
+ | <h4> <span class="mw-headline" id="Reagents_2">Reagents</span></h4> | ||
+ | <ul><li>TianGen DNA purification kit | ||
+ | </li></ul> | ||
+ | <h4> <span class="mw-headline" id="Experimental_Procedure_2">Experimental Procedure</span></h4> | ||
+ | <ol><li>Transfer PCR system (< 250uL) to 1.5mL microcentrifuge tube. Add 5 volume Buffer PD and mix. Have a quick centrifugation to remove the liquid on lid. | ||
+ | </li><li>Transfer the mixture to DNA spin column (A4) and put it in a collection tube. | ||
+ | </li><li>Centrifuge at 9000g for 1 minute and replace the collection tube with a new collection tube. | ||
+ | </li><li>To wash, add 600uL Buffer WB to the spin column (A4) and centrifuge at 7000g for 1 minute. Discard the flow-through and place the column back in the same tube. | ||
+ | </li><li>Repeat step 4. | ||
+ | </li><li>Centrifuge at 12,000g for 2 minutes. | ||
+ | </li><li>Place spin column in a clean 1.5 ml microcentrifuge tube. | ||
+ | </li><li>To elute DNA, add ddwater (>40uL) to the center of the membrane. Let the column stand for 10-15 min and centrifuge the column for 2 min at 12,000g. | ||
+ | </li><li>Add elution liquid back to spin column and centrifuge at 12,000g for 1 min. ( Inoculating ddwater at 65℃ prior would improve the elution efficiency ) | ||
+ | </li><li>Gel electrophoresis to verify the concentration of bands. | ||
+ | </li></ol> | ||
+ | <h4> <span class="mw-headline" id="Important_Notes_3">Important Notes</span></h4> | ||
+ | <ul><li>Buffer PD contains irritant compounds, thus be careful not to avoid contamination of skin. | ||
+ | </li></ul> | ||
+ | <h3> <span class="mw-headline" id="DNA_Ligation">DNA Ligation</span></h3> | ||
+ | <h4> <span class="mw-headline" id="Experimental_Procedures">Experimental Procedures</span></h4> | ||
+ | <ol><li>Set up the following reaction in a microcentrifuge tube on ice. | ||
+ | </li><li>Gently mix the reaction by pipetting up and down and microfuge briefly. | ||
+ | </li><li>For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes. | ||
+ | </li><li>For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours | ||
+ | </li><li>Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells. | ||
+ | </li></ol> | ||
+ | <h4> <span class="mw-headline" id="Ligation_System">Ligation System</span></h4> | ||
+ | <dl><dd><dl><dd><dl><dd><table border="1"> | ||
+ | <tr> | ||
+ | <td width="200"> COMPONENT | ||
+ | </td><td width="100"> 20 μl REACTION | ||
+ | </td></tr> | ||
+ | <tr> | ||
+ | <td> 10X T4 DNA Ligase Buffer | ||
+ | </td><td> 2 μl | ||
+ | </td></tr> | ||
+ | <tr> | ||
+ | <td> Vector DNA | ||
+ | </td><td> 0.025 pmol | ||
+ | </td></tr> | ||
+ | <tr> | ||
+ | <td> Insert DNA | ||
+ | </td><td> 0.076 pmol | ||
+ | </td></tr> | ||
+ | <tr> | ||
+ | <td> T4 DNA Ligase | ||
+ | </td><td> 1μl | ||
+ | </td></tr> | ||
+ | <tr> | ||
+ | <td> Nuclease-free water | ||
+ | </td><td> to 20 μl | ||
+ | </td></tr></table></dd></dl></dd></dl></dd></dl> | ||
+ | <p><br /> | ||
+ | </p> | ||
+ | <h2> <span class="mw-headline" id="h-bc"> Bacteria Culture </span></h2> | ||
+ | <h3> <span class="mw-headline" id="Strain">Strain</span></h3> | ||
+ | <ol><li>DH5a (F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15 Δ(lacZYA-argF)U169, hsdR17(rK- mK+), λ–)use for cloning | ||
+ | </li><li>BL21(DE3) (fhuA2 [lon] ompT gal (λ DE3) [dcm] ∆hsdS,λ DE3 = λ sBamHIo ∆EcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 ∆nin5) use for expressing | ||
+ | </li></ol> | ||
+ | <h3> <span class="mw-headline" id="Medium">Medium</span></h3> | ||
+ | <h4> <span class="mw-headline" id="LB_Medium">LB Medium</span></h4> | ||
+ | <dl><dd><dl><dd><dl><dd><table border="1"> | ||
+ | <tr> | ||
+ | <td width="100">NaCl | ||
+ | </td><td width="150">10g | ||
+ | </td></tr> | ||
+ | <tr> | ||
+ | <td>Tryptone | ||
+ | </td><td>10g | ||
+ | </td></tr> | ||
+ | <tr> | ||
+ | <td>Yeast Extract | ||
+ | </td><td>5 g | ||
+ | </td></tr> | ||
+ | <tr> | ||
+ | |||
+ | <td>Water | ||
+ | </td><td>up to 1000mL | ||
+ | </td></tr></table></dd></dl></dd></dl></dd></dl> | ||
+ | <h4> <span class="mw-headline" id="SOB_Medium">SOB Medium</span></h4> | ||
+ | <dl><dd><dl><dd><dl><dd><table border="1"> | ||
+ | <tr> | ||
+ | <td width="100">Tryptone | ||
+ | </td><td width="150">20g | ||
+ | </td></tr> | ||
+ | <tr> | ||
+ | <td>Yeast extract | ||
+ | </td><td>5g | ||
+ | </td></tr> | ||
+ | <tr> | ||
+ | <td>NaCl | ||
+ | </td><td>0.5g | ||
+ | </td></tr> | ||
+ | <tr> | ||
+ | <td>1M KCl | ||
+ | </td><td>2.5ml | ||
+ | </td></tr> | ||
+ | <tr> | ||
+ | <td>ddH2O | ||
+ | </td><td>up to 1000 ml | ||
+ | </td></tr></table></dd></dl></dd></dl></dd></dl> | ||
+ | <dl><dd><dl><dd>Adjust pH to 7.0 with 10N NaOH, autoclave to sterilize, and add 10 ml of 1 M MgCl2 before use | ||
+ | </dd></dl> | ||
+ | </dd></dl> | ||
+ | <h3> <span class="mw-headline" id="Antibiotics">Antibiotics</span></h3> | ||
+ | <dl><dd><dl><dd><dl><dd><table border="1"> | ||
+ | <tr> | ||
+ | <td width="70"> | ||
+ | </td><td width="150"> Storage | ||
+ | </td><td width="100">Use | ||
+ | </td></tr> | ||
+ | <tr> | ||
+ | <td width="70">Kana | ||
+ | </td><td width="150">50 mg/ml(Water) | ||
+ | </td><td>50 μg/ml | ||
+ | </td></tr> | ||
+ | <tr> | ||
+ | <td>Amp</td><td>50 mg/ml(Water)</td><td>50 μg/ml | ||
+ | </td></tr> | ||
+ | <tr> | ||
+ | <td>Tet</td><td>10 mg/ml (Alcohol)</td><td>10 μg/ml | ||
+ | </td></tr> | ||
+ | <tr> | ||
+ | <td>Cm</td><td>34 mg/ml (Alcohol)</td><td>68 μg/ml | ||
+ | </td></tr></table></dd></dl></dd></dl></dd></dl> | ||
+ | <p><br /> | ||
+ | </p> | ||
+ | <h3> <span class="mw-headline" id="Transformation">Transformation</span></h3> | ||
+ | <ol><li>Remove competent cells from freezer and allow to thaw | ||
+ | </li><li>Add 3-5 μl of DNA to the cells | ||
+ | </li><li>Incubate on ice for 30 min | ||
+ | </li><li>Heat shock the cells at 37°C for 1.5 mins | ||
+ | </li><li>Add 500 μl of SOB medium, and incubate in a shaker at 37°C for 1 hour | ||
+ | </li><li>Centrifuge for 3 min at 3,000 g, remove and keep 100 μl of the supernatant, and pour away the rest | ||
+ | </li><li>Re-suspend the pellet in the 100 μl of supernatant that was removed earlier | ||
+ | </li><li>Spread All onto one plate with corresponding antibotics, and then pour the rest onto another. Incubate the plates overnight at 37°C. | ||
+ | </li></ol> | ||
+ | <h2> <span class="mw-headline" id="h-vp">Violacein Protocols</span></h2> | ||
+ | <h3> <span class="mw-headline" id="Induction_and_Extraction">Induction and Extraction</span></h3> | ||
+ | <ol><li>Incubate the monoclonal from medium in a shaker at 37°C overnight | ||
+ | </li><li>Add 0.1% Arabinose, 1mM Ferrous ammonium sulfate and 40 μM to start induction for another 6 hours | ||
+ | </li><li>Centrifuge for 1 min at 10000rpm, remove the LB Medium | ||
+ | </li><li>Add 800 μL 10%SDS and break cells using ultrasound | ||
+ | </li><li>Add 800 μL ethyl acetate to extract violacein/deoxyviolacein | ||
+ | </li><li>Use rotovap to concentrate violacein and resuspend with methanol | ||
+ | </li></ol> | ||
+ | <h3> <span class="mw-headline" id="HPLC">HPLC</span></h3> | ||
+ | <ul><li>Column: C18, 15cm | ||
+ | </li></ul> | ||
+ | <ul><li>Mobile phase: 75% methanol, 25% ddH2O | ||
+ | </li></ul> | ||
+ | <ul><li>Injection volume: 20 μL | ||
+ | </li></ul> | ||
+ | <ul><li>Speed: 0.5 mL/min | ||
+ | </li></ul> | ||
+ | <ul><li>Detection: 230 nm | ||
+ | </li></ul> | ||
+ | <p><br /> | ||
+ | </p> | ||
+ | |||
+ | </dd></dl> | ||
</div> | </div> | ||
</div> | </div> |
Latest revision as of 03:57, 28 September 2013
Events
July 1-14
Amplified IκB fragment from the Plasmid of by Polymerase Chain Reaction(PCR).
July 15-25
Amplified GFP fragment from the Plasmid of pEGFP-N1 by Polymerase Chain Reaction(PCR).
July 25-August 1
BIT Team's member Zhou Xiaoyu visited our lab. We designed and constructed IκB-GFP fusion fragments together.
August 1-15 iGEM Lab Opening Day
Used Overlap PCR techniques to build IκB-GFP fusion fragment. Meanwhile, our team held a event which named the Opening Day of iGEM Lab for two weeks.
August 3-4
LZU-China Team member Li Xiaomeng was invited to interchange with the Peking iGEM 2013 Team.
![](https://static.igem.org/mediawiki/2013/0/0c/IMG_6928.jpg)
August 4-11 Interchange and Helping BIT
LZU-China Team member Li Xiaomeng was invited to interchange with the BIT iGEM 2013 Team. And help them to characterize and construct a part of Cr sensor.
![](https://static.igem.org/mediawiki/2013/1/18/IMG_0102.jpg)
![](https://static.igem.org/mediawiki/2013/1/1e/IMG_6942.jpg)
![](https://static.igem.org/mediawiki/2013/7/71/IMG_6943.jpg)
August 16-30
The primers synthesized two complementary single stranded DNA with NF-κB binding element. And then combined a completely DNA fragment. Used restriction enzymolysis, connection, and transformation to build NF-κB-IκB-GFP fragment.
September 1-16
Put our part into the backbone with the BioBrick prefix and suffix. And sequenced the Plasmid which will be shipped to iGEM.
Molecular Cloning Protocols
Digestion
Material
- EcoRI, BglII, PstI, XhoI, SdaI,XbaI,10X Tango buffer,10X O buffer, DNA,sterile water
Digestion temperature
- 37℃
Time period
- Plasmid: 12-16h(the higher the concentration, the longer time it takes.
- DNA fragment:12-16h(the higher the concentration, the longer time it takes.Since it is more difficult to digest DNA than plasmid, digesting DNA requires more time.
System amount
- 50ul system is preferred if there is subsequent operations. If digestion is only for identification, 20ul system is used.
- During the double digestion, the optimum buffer system which is suitable for both enzymes should be utilized. Without such shared system, digestion of two different enzymes is usually separated.
System
Enzyme 1μl 10X Tango buffer or 10X O buffer 2μl DNA ≤1μg Sterile water up to 20μl
Gel extraction
Material
- Agarose Gel DNA Extraction Kit (centrifugation)
Experimental Procedure
- Add 500uL balance liquid(BL) to the spin column(CA2). Centrifuge at 12,000 x g for 1 min at room temperature. Discard the flow-through and put the column back to the collection tube.(optional)
- Excise the DNA fragment from the agarose gel and weigh it in a clean centrifuge tube (A gel slice of 100 mg approximately equals to 100 µL).
- Add 3 volume of sol solution(PN) to the centrifuge tube and incubate the mixture at 50℃ for 10 min with mixing the tube by tapping the bottom of the tube occationally till the gel has melted completely. Cool the tube to RT.
- Transfer the mixture to an spin column (CA2) within a collection tube. Centrifuge at 12,000 x g for 1 min at room temperature. Discard the flow-through and put the column back to the collection tube.
- Add 600 µL DNA Wash Buffer (PW) to the column and centrifuge at 12,000 x g for 1 min at room temperature. Discard the flow through and put the column back to the collection tube.
- Repeat step 5.
- Put the column back to the collection tube. Centrifuge at 12,000 x g for 2 min at room temperature to discard the PW totally. Keep the lid open for several minutes.
- Put the column back to the clean collection tube. Add ddH2O to the column vacantly and keep it at room temperature for 2 minutes. Centrifuge at 12,000 x g for 2 min at room temperature to collect the DNA solution.
- Transfer the eluted solution to CA2 and keep it at room temperature for 2minutes. Centrifuge at 12,000 x g for 2 min at room temperature.
- Gel electrophoresis to verify the correctness and concentration of bands.
Important Notes
- If the balance liquid turns turbid, incubate it at 37℃ for several minutes.
- When cutting the gel, keep the UV irradiation time period as short as possible, otherwise DNA would be damaged.
- For those DNA fragment shorter than 100bp or larger than 10kb, the sol solution volume should be increased as well as the time period for adsorption and elution.
- Recovery depends on the initial DNA amount and elution volume. The less the DNA, the less the elution volume, the smaller of recovery.
PCR
Reagents
- Pfu enzymes
- Tap enzymes
Experiment system
- Experiment system with Pfu enzymes (Mostly 50uL)
- Experiment system with taq enzymes:
- Reaction temperature:
Two-step method: Predenature 94℃,2min Denature and extension { 94℃ 30sec 72℃ 60sec/kb } × 25-45 cycles 4℃
Three-step method: Predenature 94℃,2min Denature,annealing and extension 94℃ 30sec { (Tm-5)℃ 30sec 72℃ 60sec/kb } × 25-45 cycles 4℃
Important Notes
- Pfu enzymes should be operated in ice.
- Two-step method is preferred if primer Tm is relatively high.
- 1.2% agarose gel is used to separate 1kb-5kb DNA; 0.8% agarose gel is used to separate 3k-10k DNA; 2.5-3% agarose gel is used to separate DNA longer than 100bp.
- PCR reaction system varies from 20uL to 100uL. Under most cases, 20uL system is preferred as long as PCR is for identification only. However, when subsequent experiments such as tailing, digestion which require certain amount of DNA are necessary, 50uL system is usually preferred.
- Pfu enzymes are preferred if the purpose of PCR is for gene amplification. At the same time, PCR for identification or tailing usually requires taq enzymes.
PCR Purification
Reagents
- TianGen DNA purification kit
Experimental Procedure
- Transfer PCR system (< 250uL) to 1.5mL microcentrifuge tube. Add 5 volume Buffer PD and mix. Have a quick centrifugation to remove the liquid on lid.
- Transfer the mixture to DNA spin column (A4) and put it in a collection tube.
- Centrifuge at 9000g for 1 minute and replace the collection tube with a new collection tube.
- To wash, add 600uL Buffer WB to the spin column (A4) and centrifuge at 7000g for 1 minute. Discard the flow-through and place the column back in the same tube.
- Repeat step 4.
- Centrifuge at 12,000g for 2 minutes.
- Place spin column in a clean 1.5 ml microcentrifuge tube.
- To elute DNA, add ddwater (>40uL) to the center of the membrane. Let the column stand for 10-15 min and centrifuge the column for 2 min at 12,000g.
- Add elution liquid back to spin column and centrifuge at 12,000g for 1 min. ( Inoculating ddwater at 65℃ prior would improve the elution efficiency )
- Gel electrophoresis to verify the concentration of bands.
Important Notes
- Buffer PD contains irritant compounds, thus be careful not to avoid contamination of skin.
DNA Ligation
Experimental Procedures
- Set up the following reaction in a microcentrifuge tube on ice.
- Gently mix the reaction by pipetting up and down and microfuge briefly.
- For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.
- For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours
- Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.
Ligation System
COMPONENT 20 μl REACTION 10X T4 DNA Ligase Buffer 2 μl Vector DNA 0.025 pmol Insert DNA 0.076 pmol T4 DNA Ligase 1μl Nuclease-free water to 20 μl
Bacteria Culture
Strain
- DH5a (F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15 Δ(lacZYA-argF)U169, hsdR17(rK- mK+), λ–)use for cloning
- BL21(DE3) (fhuA2 [lon] ompT gal (λ DE3) [dcm] ∆hsdS,λ DE3 = λ sBamHIo ∆EcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 ∆nin5) use for expressing
Medium
LB Medium
NaCl 10g Tryptone 10g Yeast Extract 5 g Water up to 1000mL
SOB Medium
Tryptone 20g Yeast extract 5g NaCl 0.5g 1M KCl 2.5ml ddH2O up to 1000 ml
- Adjust pH to 7.0 with 10N NaOH, autoclave to sterilize, and add 10 ml of 1 M MgCl2 before use
Antibiotics
Storage Use Kana 50 mg/ml(Water) 50 μg/ml Amp 50 mg/ml(Water) 50 μg/ml Tet 10 mg/ml (Alcohol) 10 μg/ml Cm 34 mg/ml (Alcohol) 68 μg/ml
Transformation
- Remove competent cells from freezer and allow to thaw
- Add 3-5 μl of DNA to the cells
- Incubate on ice for 30 min
- Heat shock the cells at 37°C for 1.5 mins
- Add 500 μl of SOB medium, and incubate in a shaker at 37°C for 1 hour
- Centrifuge for 3 min at 3,000 g, remove and keep 100 μl of the supernatant, and pour away the rest
- Re-suspend the pellet in the 100 μl of supernatant that was removed earlier
- Spread All onto one plate with corresponding antibotics, and then pour the rest onto another. Incubate the plates overnight at 37°C.
Violacein Protocols
Induction and Extraction
- Incubate the monoclonal from medium in a shaker at 37°C overnight
- Add 0.1% Arabinose, 1mM Ferrous ammonium sulfate and 40 μM to start induction for another 6 hours
- Centrifuge for 1 min at 10000rpm, remove the LB Medium
- Add 800 μL 10%SDS and break cells using ultrasound
- Add 800 μL ethyl acetate to extract violacein/deoxyviolacein
- Use rotovap to concentrate violacein and resuspend with methanol
HPLC
- Column: C18, 15cm
- Mobile phase: 75% methanol, 25% ddH2O
- Injection volume: 20 μL
- Speed: 0.5 mL/min
- Detection: 230 nm
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