Team:UESTC-China/Protocol

From 2014.igem.org

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       <h1 class="textEditingTitle" style="width: 600px">E. coli Calcium Chloride competent cell protocol</br>
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       <h1 class="textEditingTitle" style="width: 1100px">E. coli Calcium Chloride competent cell protocol</br>
         </h1><p class="textEditingstyle">1)Streak E.coli cells (DH5a) on an LB plate; (BL21(DE3)LysS cells on LB plate+34 mg/ml chloramphenicol)<br>
         </h1><p class="textEditingstyle">1)Streak E.coli cells (DH5a) on an LB plate; (BL21(DE3)LysS cells on LB plate+34 mg/ml chloramphenicol)<br>
           2) Allow cells to grow at 37℃ overnight<br>
           2) Allow cells to grow at 37℃ overnight<br>
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           18) Count the number of colonies.<br>
           18) Count the number of colonies.<br>
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<h1 class="textEditingTitle" style="width: 600px">Agrobacterium tumefacien EHA105 Mediated Transformation with freeze-thawing steps</br></h1>
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<h1 class="textEditingTitle" style="width: 1100px">Agrobacterium tumefacien EHA105 Mediated Transformation with freeze-thawing steps</br></h1>
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(1)Have 0.5~1  g plasmid DNA into 100  L competent Cells,on ice for 30 min;<br>
(1)Have 0.5~1  g plasmid DNA into 100  L competent Cells,on ice for 30 min;<br>
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(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>
(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>
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<h1 class="textEditingTitle" style="width:600px">Agrobacterium-mediated genetic transformation to tobacco </br></h1>
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<h1 class="textEditingTitle" style="width:1100px">Agrobacterium-mediated genetic transformation to tobacco </br></h1>
         <p class="textEditingstyle">Tobacco was transformed essentially by following the leaf disk co-cultivation protocol of Horsch et al[1]. 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 (Murashige and Skoog 1962 )  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 class="textEditingstyle">Tobacco was transformed essentially by following the leaf disk co-cultivation protocol of Horsch et al[1]. 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 (Murashige and Skoog 1962 )  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).
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<h1 class="textEditingTitle" style="width: 600px">Detection method </br></h1>
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<h1 class="textEditingTitle" style="width: 1100px">Detection method </br></h1>
         <p class="textEditingstyle">1)Molecular identification<br>
         <p class="textEditingstyle">1)Molecular identification<br>

Revision as of 08:32, 16 October 2014

UESTC-China