Team:UESTC-China/Protocol
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<div class="tableEditing"> | <div class="tableEditing"> | ||
- | <div id="SectionTitles1" style="width:1100px;"> | + | <h1 style="color:#1b1b1b; position:relative; left:25px; padding:15 5px; font-size:40px; font-family: calibri, arial, helvetica, sans-serif; font-weight: bold;font-style: Italic; text-align:center; width:1140px;">Protocol</h1> |
+ | <div id="SectionTitles1" style="width:1100px;">Fragments Ligation | ||
</h1> | </h1> | ||
- | <table border="1" id="BiosensorsTable" cellspacing="0";> | + | <table border="1" id="BiosensorsTable" cellspacing="0"; style="margin-left: 50px;"> |
<tr> | <tr> | ||
<th class="top">Component</th> | <th class="top">Component</th> | ||
- | <th class="top">20µl Reaction</th> | + | <th class="top">20µl Reaction </th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td class="mid"> | + | <td class="mid">Vector DNA</td> |
- | <td class="mid"> | + | <td class="mid">50ng</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td class="mid">Insert DNA </td> | + | <td class="mid">Insert DNA</td> |
- | <td class="mid"> the moles of insert DNA to vector DNA is 5:1</td> | + | <td class="mid"> the moles rate of insert DNA to vector DNA is 5:1</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td class="mid"> | + | <td class="mid">10X T4 DNA ligase buffer</td> |
- | <td class="mid"> | + | <td class="mid">2µl</td> |
+ | </tr> | ||
+ | <tr> | ||
+ | <td class="mid">T4 DNA ligase</td> | ||
+ | <td class="mid">1µl</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td class="mid">ddH2O</td> | <td class="mid">ddH2O</td> | ||
- | <td class="mid"> | + | <td class="mid">to 20µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td class="mid">Incubate at | + | <td class="mid">Incubate at 25℃ for 12 hours</td> |
<td class="mid"></td> | <td class="mid"></td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
+ | |||
+ | |||
</div> | </div> | ||
- | <div id="SectionTitles2" style="width:1100px;"> | + | <div id="SectionTitles2" style="width:1100px;">Gibson Assembly |
</h1> | </h1> | ||
- | <table border="1" id="BiosensorsTable" cellspacing="0";> | + | |
+ | |||
+ | |||
+ | |||
+ | |||
+ | <table border="1" id="BiosensorsTable" cellspacing="0"; style="margin-left: 50px;"> | ||
<tr> | <tr> | ||
<th class="top">Component</th> | <th class="top">Component</th> | ||
- | <th class="top">20µl Reaction </th> | + | <th class="top">20µl Reaction</th> |
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td class="mid"> | + | <td class="mid">1.33X Master Mix</td> |
- | <td class="mid"> | + | <td class="mid"> 15µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td class="mid">Insert DNA</td> | + | <td class="mid">Insert DNA </td> |
- | <td class="mid"> the moles of insert DNA to vector DNA is 5:1</td> | + | <td class="mid"> the moles rate of insert DNA to vector DNA is 5:1</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td class="mid"> | + | <td class="mid">Vector DNA</td> |
- | <td class="mid"> | + | <td class="mid"> 50ng</td> |
- | + | ||
- | + | ||
- | + | ||
- | + | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td class="mid">ddH2O</td> | <td class="mid">ddH2O</td> | ||
- | <td class="mid"> | + | <td class="mid"> To 20µl</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td class="mid">Incubate at | + | <td class="mid">Incubate at 50℃ for 1 hour</td> |
<td class="mid"></td> | <td class="mid"></td> | ||
</tr> | </tr> | ||
</table> | </table> | ||
+ | |||
+ | |||
+ | |||
+ | |||
</div> | </div> | ||
- | <div id="SectionTitles3" style="width:1100px;"> | + | <div id="SectionTitles3" style="width:1100px;">Fragment Digestion |
</h1> | </h1> | ||
- | <table border="1" id="BiosensorsTable" cellspacing="0";> | + | <table border="1" id="BiosensorsTable" cellspacing="0"; style="margin-left: 50px;"> |
<tr> | <tr> | ||
<th class="top">Component</th> | <th class="top">Component</th> | ||
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<tr> | <tr> | ||
<td class="mid">DNA</td> | <td class="mid">DNA</td> | ||
- | <td class="mid">1- | + | <td class="mid">1-2µg</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
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</table> | </table> | ||
</div> | </div> | ||
- | <div id="SectionTitles4" style="width:1100px;">PCR Protocol for KOD-Plus-Neo | + | <div id="SectionTitles4" style="width:1100px;">PCR Amplication Protocol for KOD-Plus-Neo |
</h1> | </h1> | ||
- | <table border="1" id="BiosensorsTable" cellspacing="0";> | + | <table border="1" id="BiosensorsTable" cellspacing="0"; style="margin-left: 50px;"> |
<tr> | <tr> | ||
<th class="top">Component</th> | <th class="top">Component</th> | ||
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<tr> | <tr> | ||
<td class="mid">10mM dNTPs</td> | <td class="mid">10mM dNTPs</td> | ||
- | <td class="mid"> | + | <td class="mid">5µg</td> |
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td class="mid"> | + | <td class="mid">10µM forward primer</td> |
<td class="mid">1µl</td> | <td class="mid">1µl</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td class="mid"> | + | <td class="mid">10µM reverse primer</td> |
<td class="mid">1µl</td> | <td class="mid">1µl</td> | ||
</tr> | </tr> | ||
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</div> | </div> | ||
<div id="SectionTitles5" style="width:1100px;"> | <div id="SectionTitles5" style="width:1100px;"> | ||
- | <table border="1" id="BiosensorsTable" cellspacing="0";> | + | <table border="1" id="BiosensorsTable" cellspacing="0"; style="margin-left: 50px;"> |
<tr> | <tr> | ||
<th class="top"></th> | <th class="top"></th> | ||
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</table> | </table> | ||
</div> | </div> | ||
- | <div id="SectionTitles6" style="width:1100px;">Fusion PCR | + | <div id="SectionTitles6" style="width:1100px;">Fusion PCR Amplication |
</h1> | </h1> | ||
<p id="underTitle">Each template will be diluted by the number of moles into 2-10ng/µl.</p> | <p id="underTitle">Each template will be diluted by the number of moles into 2-10ng/µl.</p> | ||
- | <div id="fusionArea" ><table border="1" id="BiosensorsTable" cellspacing="0";> | + | <div id="fusionArea" ><table border="1" id="BiosensorsTable" cellspacing="0"; style="margin-left: 50px;"> |
<tr> | <tr> | ||
<th class="top">Component</th> | <th class="top">Component</th> | ||
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</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td class="mid"> | + | <td class="mid">10µM forward primer</td> |
<td class="mid">1µl</td> | <td class="mid">1µl</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td class="mid"> | + | <td class="mid">10µM reverse primer</td> |
<td class="mid">1µl</td> | <td class="mid">1µl</td> | ||
</tr> | </tr> | ||
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</div> | </div> | ||
<div id="SectionTitles7" style="width:1100px;"> | <div id="SectionTitles7" style="width:1100px;"> | ||
- | <table border="1" id="BiosensorsTable" cellspacing="0";> | + | <table border="1" id="BiosensorsTable" cellspacing="0"; style="margin-left: 50px;"> |
<tr> | <tr> | ||
<th class="top"></th> | <th class="top"></th> | ||
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</table> | </table> | ||
</div> | </div> | ||
- | <div id="SectionTitles8" style="width:1100px;">Colony PCR | + | <div id="SectionTitles8" style="width:1100px;">Colony PCR |
</h1> | </h1> | ||
- | <table border="1" id="BiosensorsTable" cellspacing="0";> | + | <table border="1" id="BiosensorsTable" cellspacing="0"; style="margin-left: 50px;"> |
<tr> | <tr> | ||
<th class="top">Component</th> | <th class="top">Component</th> | ||
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</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td class="mid"> | + | <td class="mid">10µM forward primer</td> |
<td class="mid">0.5µl</td> | <td class="mid">0.5µl</td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
- | <td class="mid"> | + | <td class="mid">10µM reverse primer</td> |
<td class="mid">0.5µl</td> | <td class="mid">0.5µl</td> | ||
</tr> | </tr> | ||
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</div> | </div> | ||
<div id="SectionTitles9" style="width:1100px;"> | <div id="SectionTitles9" style="width:1100px;"> | ||
- | <table border="1" id="BiosensorsTable" cellspacing="0";> | + | <table border="1" id="BiosensorsTable" cellspacing="0"; style="margin-left: 50px;"> |
<tr> | <tr> | ||
<th class="top"></th> | <th class="top"></th> | ||
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</div> | </div> | ||
<div class="textEditingArea" > | <div class="textEditingArea" > | ||
- | <h1 class="textEditingTitle" style="width: 1100px">E. coli | + | |
- | </h1><p class="textEditingstyle">1)Streak E.coli cells (DH5α) on an LB plate, (BL21(DE3)LysS cells on LB plate + 25 mg/ml chloramphenicol);<br> | + | <h1 class="textEditingTitle" style="width: 1100px">E. coli Transformation</br> |
+ | </h1><p class="textEditingstyle">1)Streak <i>E.coli</i> cells (DH5α) on an LB plate, (BL21 (DE3) LysS cells on LB plate + 25 mg/ml chloramphenicol);<br> | ||
2) Allow cells to grow at 37℃ overnight;<br> | 2) Allow cells to grow at 37℃ overnight;<br> | ||
3)Place one colony in 10 ml LB media (+ antibiotic selection if necessary), grow overnight at 37℃;<br> | 3)Place one colony in 10 ml LB media (+ antibiotic selection if necessary), grow overnight at 37℃;<br> | ||
- | 4) Take 2 ml LB media and save for blank, | + | 4) Take 2 ml LB media and save for blank, transfer 5 ml overnight DH5α culture into 500 ml LB media in 3 L flask;<br> |
5) Allow cell to grow at 37℃ (250 rpm), until OD600= 0.4 (~2-3 hours);<br> | 5) Allow cell to grow at 37℃ (250 rpm), until OD600= 0.4 (~2-3 hours);<br> | ||
6) Transfer cells to 2 centrifuge bottles (250 ml), and place cells on ice for 20 min;<br> | 6) Transfer cells to 2 centrifuge bottles (250 ml), and place cells on ice for 20 min;<br> | ||
- | 7) Centrifuge cells in at | + | 7) Centrifuge cells in at 4℃ for 10 min at 3,000 g and subsequent resuspension may be done in the same bottle. Cells must remain cold for the rest of the procedure: Transport tubes on ice and resuspend on ice in the cold room;<br> |
8) Pour off media and resuspend cells in 30 ml of cold 0.1 M CaCl2. Transfer the suspended cells into 50 ml polypropylene tubes, and incubate on ice for 30 min;<br> | 8) Pour off media and resuspend cells in 30 ml of cold 0.1 M CaCl2. Transfer the suspended cells into 50 ml polypropylene tubes, and incubate on ice for 30 min;<br> | ||
- | 9) Centrifuge cells at | + | 9) Centrifuge cells at 40℃ for 10 min at 3,000 g;<br> |
- | 10) Pour supernatant and resuspend cells (by pipetting) in 8 ml cold 0.1M CaCl2 containing 15% glycerol Transfer 140 ml into (1.5 ml) | + | 10) Pour supernatant and resuspend cells (by pipetting) in 8 ml cold 0.1M CaCl2 containing 15% glycerol Transfer 140 ml into (1.5 ml).Eppendorf tubes placed on ice. Freeze the cells in liquid nitrogen. Cells stored at -80℃ can be used for transformation for up to ~6 months;<br> |
11) Add 10 to 40 ng (10 to 25 ml volume) of DNA to 250 ml of competent cells in step;<br> | 11) Add 10 to 40 ng (10 to 25 ml volume) of DNA to 250 ml of competent cells in step;<br> | ||
12) Incubate the mixture on ice for 30 minutes;<br> | 12) Incubate the mixture on ice for 30 minutes;<br> | ||
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15) Incubate on ice for 2 minutes;<br> | 15) Incubate on ice for 2 minutes;<br> | ||
16) Spread the appropriate quantity of cells (50 to 100 ml) on selective media. Store the remaining cells at 4℃.<br> | 16) Spread the appropriate quantity of cells (50 to 100 ml) on selective media. Store the remaining cells at 4℃.<br> | ||
- | (A) E. coli cells from the control tube without DNA in step 12 above are plated on selective medium and nonselective medium. The first plating ensures that the selective medium is working properly since no growth should be observed. The second plating provides the number of viable cells in the absence of selective medium. <br> | + | (A) <i>E. coli</i> cells from the control tube without DNA in step 12 above are plated on selective medium and nonselective medium. The first plating ensures that the selective medium is working properly since no growth should be observed. The second plating provides the number of viable cells in the absence of selective medium. <br> |
- | (B) E. coli cells being tested for competency are plated on LB agar containing ampicillin (50 mg/ml final concentration) to ensure that the transformation efficiency has not decreased over time due to storage.<br> | + | (B) <i>E. coli</i> cells being tested for competency are plated on LB agar containing ampicillin (50 mg/ml final concentration) to ensure that the transformation efficiency has not decreased over time due to storage.<br> |
- | 17) Incubate all plates overnight at 37℃ | + | 17) Incubate all plates overnight at 37℃.<br> |
18) Count the number of colonies.<br> | 18) Count the number of colonies.<br> | ||
</p> | </p> | ||
- | <h1 class="textEditingTitle" style="width: 1100px"> | + | <h1 class="textEditingTitle" style="width: 1100px">A.tume Transformation</br></h1> |
<p class="textEditingstyle"> | <p class="textEditingstyle"> | ||
- | + | 1)Have 0.5~1 g plasmid DNA into 100 L competent cells, on ice for 30 min;<br> | |
- | + | 2)Frozen in liquid nitrogen for 5 min, grow at 37 C for 5 min, on ice for 2 min;<br> | |
- | + | 3)Pour in 1000 LYEB, 200 rpm, grow at 28℃ for 2~3 h;<br> | |
- | + | 4)6000rpm,2min. Suspend collected bacteria with 100µl YEB and evenly coat that at a YEB medium. (125 mg/L Sm or 50 mg/L rif, and 50 mg/L Kan included);<br> | |
- | + | 5)Grow upside down at 28℃, for 48h;<br> | |
- | + | 6)Pick positive clones and grow them in LB medium with antibiotic at 28℃ for 48h;<br> | |
- | + | 7)Inject to LB medium in flasks by the day of transformation, in the rate of 1:50. Grow to OD600 = 0.5. Ready to infect tobacco leaf discs.<br> | |
</p> | </p> | ||
- | <h1 class="textEditingTitle" style="width:1100px">Agrobacterium-mediated | + | <h1 class="textEditingTitle" style="width:1100px">Agrobacterium-mediated Tobacco Transformation</br></h1> |
- | <p class="textEditingstyle">Tobacco was transformed essentially by following the leaf disk co-cultivation protocol of | + | <p class="textEditingstyle">Tobacco was transformed essentially by following the leaf disk co-cultivation protocol of <i>(McCormick et al., 1986)</i>. Co-cultivation was initiated by dipping leaf disks in an Agrobacterium suspension, blotting them on sterile tissue paper, and incubating them for 2 d on MS medium <i>(Murashige and Skoog 1962)</i> containing naphthalene acetic acid (NAA 0.1mg/L), 6-Benzylaminopurine (6-BA, 2.0mg/L). Cefotaxime sodium (Cef) was included in the medium (500mg/L) to inhibit Agrobacterium growth. The leaf disks were then transferred onto a medium containing antibiotics for transgenic plant selection(kanamycin, 50 mg/L), and NAA (0.1 mg/L), 6-BA (2.0 mg/L), Cef (500mg/L). And incubate them for 1 month on the medium above. At last, cut off the bud from the callus, put the buds into the mudium containing NAA (0.1mg/L), Cef (500mg/L) and kanamycin (25 mg/L). |
</p> | </p> | ||
<h1 class="textEditingTitle" style="width: 1100px">Detection Method </br></h1> | <h1 class="textEditingTitle" style="width: 1100px">Detection Method </br></h1> | ||
- | <p class="textEditingstyle">1)Molecular | + | <p class="textEditingstyle">1)Molecular Identification<br> |
- | Extract | + | Extract genomic DNA from leaves of tobacco seedlings. Using specific primers to amplify the target gene. Extract leaf mRNA from leaves of grown tobacco which DNA testing was positive. Detected by the method of RT-PCR whether target gene is expressed.<br> |
2)Phenotype testing<br> | 2)Phenotype testing<br> | ||
</p> | </p> | ||
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<td class="lastmid">Qualitative detection</td> | <td class="lastmid">Qualitative detection</td> | ||
- | <td class="lastmid">Have 6 positive | + | <td class="lastmid">Have 6 positive plants from every transgenic line (about 8 leaves age) , and 6 wild-type seedings with the same growth equally distributed into three 650ml culture bottles. Treat with 10µl 37% formaldehyde for two weeks. </td> |
<td class="mid"></td> | <td class="mid"></td> | ||
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<td class="lastmid"><br>Quantitative detection</td> | <td class="lastmid"><br>Quantitative detection</td> | ||
- | <td class="lastmid"><br> Have 4 positive | + | <td class="lastmid"><br> Have 4 positive plants from every transgenic line (about 8 leaves age) in a 650ml culture bottle. Treat with 10µl 37% formaldehyde for 3 weeks. Using a formaldehyde detector (Gastec Passive Dositube, 91D, Ayase, Kanagawa, Japan) was set in the hole to detect gaseous formaldehyde. Three and a half hours later, the measurement was stopped and the results were photographed <i>(Chen et al., 2010)</i>.</td> |
- | <td class="lastmid"><br>Put 4 wild -type seedlings with the same growth of seedlings in experimental group into 650ml culture | + | <td class="lastmid"><br>Put 4 wild -type seedlings with the same growth of seedlings in experimental group into 650ml culture bottle,with same processing as the case of the experimental group.</td> |
</tr> | </tr> | ||
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<p id="references"><b>References</b><br> | <p id="references"><b>References</b><br> | ||
- | McCormick, S., J. Niedermeyer, J. Fry, A. Barnason, R. Horsch and R. Fraley (1986). "Leaf disc transformation of cultivated tomato (L. esculentum) using Agrobacterium tumefaciens." | + | <i>McCormick, S., J. Niedermeyer, J. Fry, A. Barnason, R. Horsch and R. Fraley (1986). "Leaf disc transformation of cultivated tomato (L. esculentum) using Agrobacterium tumefaciens."Plant Cell Rep 5(2): 81-84.<br> |
- | Chen, L. M., H. Yurimoto, K. Z. Li, I. Orita, M. Akita, N. Kato, Y. Sakai and K. Izui (2010). "Assimilation of formaldehyde in transgenic plants due to the introduction of the bacterial ribulose monophosphate pathway genes." | + | Chen, L. M., H. Yurimoto, K. Z. Li, I. Orita, M. Akita, N. Kato, Y. Sakai and K. Izui (2010). "Assimilation of formaldehyde in transgenic plants due to the introduction of the bacterial ribulose monophosphate pathway genes." Biosci Biotechnol Biochem</i> 74(3): 627-635.</i><br> |
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Latest revision as of 03:29, 18 October 2014