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| | {{:Team:Tianjin/Templates/MaterialTheme|}} | | {{:Team:Tianjin/Templates/MaterialTheme|}} |
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| | + | {{:Team:Tianjin/Templates/AddCSS|:Team:Tianjin/Note/R-R/camarts.css}} |
| | {{:Team:Tianjin/Templates/AddCSS|:Team:Tianjin/Experiment/css/font.css}} | | {{:Team:Tianjin/Templates/AddCSS|:Team:Tianjin/Experiment/css/font.css}} |
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| | <html lang="en"> | | <html lang="en"> |
| | + | <style> |
| | + | #Notes{ |
| | + | color: inherit; |
| | + | background-color: rgba(255, 255, 255, 0.1); |
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| | + | #content article{margin:0;padding:0;} |
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| | <head> | | <head> |
| | <meta charset="utf-8" /> | | <meta charset="utf-8" /> |
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| − | <li>
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| − | <h1 style="font-size:135%">Modification of Cyanobacteria</h1><br/>
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| − | <b><h1 style="font-size:108%"> Jul.31th</b></h1>
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| − | <div style="padding-left:32px;">1.Cultivation: 30µL mother liquid of Synechococcus sp PCC 7942 (wild type)were cultivated in 10 mL BG-11-media at 30°C and 140rpm.<br/>
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| − | 2.The genome DNA of 7942 was extracted using a Genomic DNA Isolation Kit.</div><br/>
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| | </li> | | </li> |
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| − | <li>
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| − | <h1 style="font-size:135%">Modification of Cyanobacteria</h1><br/>
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| − | <div style="padding-left:32px;">Synechococcus sp PCC 7942 had no signals of life.</div><br/>
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| | </div> | | </div> |
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| | </li> | | </li> |
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| − | <li>
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| − | <h1 style="font-size:135%">Modification of Cyanobacteria</h1><br/>
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| − | <b><h1 style="font-size:108%"> Aug.29th</b></h1>
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| − | <div style="padding-left:32px;">1.pMV-G19 and pMV-G15 containing our target genes were received.<br/>
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| − | 2.Colonies were used to inoculate overnight cultures.
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| − | </div>
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| − | <br/>
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| − | <b><h1 style="font-size:108%"> Aug.30th</b></h1>
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| − | <div style="padding-left:32px;">1.Plasmids were isolated using a miniprep kit.<br/>
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| − | 2.Amplification of <i>19</i> and <i>15</i> with Q5 High-Fidelity DNA Polymerase out of E.coli.<br/>
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| − | <i>19</i> amplification at 65.0°C with 19.rev/fwd primes.<br/>
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| − | PCR worked, positive control worked, no amplification of <i>19</i>.<br/>
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| − | <i>15</i> amplification at 65.0°C with 15.rev/fwd primes.<br/>
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| − | PCR worked, positive control worked, no amplification of <i>15</i>.<br/>
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| − | 3.A product length of 900 bp was expected. The gel shows that most plasmids seem to be correct.<br/>
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| − | This result was confirmed by sequencing.<br/>
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| − | 4.The fragments of <i>19</i> and <i>15</i> were purified with PCR Purification Kit.<br/>
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| − | </div>
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| − | <br/>
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| − |
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| − | <b><h1 style="font-size:108%"> Aug.31th</b></h1>
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| − | <div style="padding-left:32px;">1.Ligation of <i>15</i> with <i>19</i>.<br/>
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| − | 2.We add a single 3`-adenine overhang to each end of the fragment of <i>19</i> and then purified with DNA Purification Kit.<br/>
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| − | 3.<i>19</i> was ligated into T vectors via TA clone and transformed into E.coli via heat shock.<br/>
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| − | </div>
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| − | <br/>
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| − | <b><h1 style="font-size:108%"> Sep.1th</b></h1>
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| − | <div style="padding-left:32px;">1.A colony PCR was performed with five colonies.<br/>
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| − | 2.Gel electrophoresis showed that 5 colonies were positive for insertion of <i>19</i>.<br/>
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| − | 3.Two of these colonies containing <i>19</i> were used to inoculate overnight cultures.<br/>
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| − | 4.<i>15</i> gene fragment was phosphorylated.<br/>
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| − | </div>
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| − | <br/>
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| − | <b><h1 style="font-size:108%"> Sep.2th</b></h1>
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| − | <div style="padding-left:32px;">1.Plasmids containing T vector with <i>19</i>(pT-19)were isolated using a miniprep kit.<br/>
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| − | 2.Mono-restriction digest of pT-19 with stu I. <br/>
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| − | 3.The enzyme-digested product was dephosphorylation.<br/>
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| − | 4.Dephosphorylated plasmid and phosphorylated gene <i>15</i> were connected.<br/>
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| − | 5.Ligation product was transformed into E.coli via heat shock.<br/>
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| − | </div>
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| − | <br/>
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| − | <b><h1 style="font-size:108%"> Sep.3th</b></h1>
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| − | <div style="padding-left:32px;">1.A colony PCR was performed with twelve colonies.<br/>
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| − | 2.Gel electrophoresis showed that 2 colonies were positive for connecting the plasmid and gene fragment.<br/>
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| − | 3.These two colonies were used to inoculate overnight cultures.<br/>
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| − | </div>
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| − | <br/>
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| | </li> | | </li> |
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| − | <li>
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| − | <h1 style="font-size:135%">Modification of Cyanobacteria</h1><br/>
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| − |
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| − | <b><h1 style="font-size:108%"> Sep.4th</b></h1>
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| − | <div style="padding-left:32px;">1.Plasmids with correct sequence of <i>19-15</i> were isolated using a miniprep kit.<br/>
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| − | 2.Mono-restriction digest of pT-19-15 with Nru I.<br/>
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| − | 3.The enzyme-digested product was dephosphorylation.<br/>
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| − | </div>
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| − | <br/>
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| − | <b><h1 style="font-size:108%"> Sep.6th</b></h1>
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| − | <div style="padding-left:32px;">Colonies containing gene <i>13</i> were used to inoculate overnight cultures.
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| − | <br/>
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| − | </div>
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| − | <br/>
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| − | <b><h1 style="font-size:108%"> Sep.7th</b></h1>
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| − | <div style="padding-left:32px;">1.Plasmids were isolated using a miniprep kit.<br/>
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| − | 2.Amplification of <i>13</i> with Q5 High-Fidelity DNA Polymerase out of E.coli <br/>
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| − | 3.<i>13</i> amplification at 65.0°C with 13.rev/fwd primes<br/>
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| − | PCR worked, positive control worked, no amplification of <i>13</i><br/>
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| − | 4.The fragments of <i>13</i> were purified with PCR Purification Kit.<br/>
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| − | </div>
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| − | <br/>
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| − | <b><h1 style="font-size:108%"> Sep.8th</b></h1>
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| − | <div style="padding-left:32px;"><i>13</i> gene fragment was phosphorylated.<br/>
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| − | </div>
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| − | <br/>
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| − | <b><h1 style="font-size:108%"> Sep.9th</b></h1>
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| − | <div style="padding-left:32px;">1.Insertion of Ni promoter and ligation of <i>13-19-15</i><br/>
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| − | 2.Ni inducible promoter was ligated into pCPC-3301 vector.<br/>
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| − | 3.Phosphorylated <i>13</i> was ligated into pT-19-15 and transformed into E.coli via heat shock.<br/>
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| − | 4.Single colonies were obtained by plating.<br/>
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| − | </div>
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| − | <br/>
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| − |
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| − | <b><h1 style="font-size:108%"> Sep.10th</b></h1>
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| − | <div style="padding-left:32px;">
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| − | 1.A colony PCR of Ni inducible promoter(pCPC-3301-Ni) was performed with 12 colonies.<br/>
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| − | Two of the successful ones were used to inoculate overnight cultures.<br/>
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| − | 2.A colony PCR of pT-13-19-15 was performed with 7 colonies.<br/>
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| − | Two of the successful ones were used to inoculate overnight cultures.<br/>
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| − | </div>
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| − | <br/>
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| − | <li>
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| − | <h1 style="font-size:135%">Modification of Cyanobacteria</h1><br/>
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| − | <b><h1 style="font-size:108%"> Sep.11th</b></h1>
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| − | <div style="padding-left:32px;">Two kinds of plasmids were isolated using a miniprep kit.<br/>
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| − | </div>
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| − | <br/>
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| − | <b><h1 style="font-size:108%"> Sep.12th</b></h1>
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| − | <div style="padding-left:32px;">Two kinds of plasmids were isolated using a miniprep kit.
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| − | <br/>
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| − | </div>
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| − | <br/>
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| − | <b><h1 style="font-size:108%"> Sep.14th</b></h1>
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| − | <div style="padding-left:32px;">The sequencing results for both of them were error.<br/>
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| − | </div>
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| − | <br/>
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| − | <b><h1 style="font-size:108%"> Sep.15th</b></h1>
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| − | <div style="padding-left:32px;">1.Colonies containing Ni inducible promoter were used to inoculate overnight cultures.<br/>
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| − | 2.PCR was performed to check if the gene fragments were ligated correctly.<br/>
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| − | 13_ fwd and 15_rev on pT-13-19-15<br/>
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| − | 3.Gel electrophoresis showed that it failed.<br/>
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| − | </div>
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| − | <br/>
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| − |
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| − |
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| − | <b><h1 style="font-size:108%"> Sep.16th</b></h1>
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| − | <div style="padding-left:32px;">Plasmids pCPC-3031-Ni were isolated using a miniprep kit.<br/>
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| − | </div>
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| − | <br/>
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| − |
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| − | <b><h1 style="font-size:108%"> Sep.17th</b></h1>
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| − | <div style="padding-left:32px;">
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| − | 1.Several PCRs were performed to check if the gene fragments were ligated correctly.<br/>
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| − | 13_fwd and 13_rev on pT-13-19-15<br/>
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| − | 19_fwd and 19_rev on pT-13-19-15<br/>
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| − | 15_fwd and 15_rev on pT-13-19-15<br/>
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| − | 13_fwd and 19_rev on pT-13-19-15<br/>
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| − | 19_fwd and 15_rev on pT-13-19-15<br/>
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| − | 13_ wd and 15_rev on pT-13-19-15<br/>
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| − | <b>The fourth and sixth ones were not successful.</b><br/>
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| − | 2.<b>Repetition:</b> Phosphorylated <i>13</i> was ligated into pT-19-15 and transformed into E.coli via heat shock.<br/>
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| − | </div>
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| − | <br/>
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| − | </li>
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| | </div> | | </div> |
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| | <br/> | | <br/> |
| | </li> | | </li> |
| − |
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| − | <li>
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| − | <h1 style="font-size:135%">Modification of Cyanobacteria</h1><br/>
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| − |
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| − | <b><h1 style="font-size:108%"> Sep.18th</b></h1>
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| − | <div style="padding-left:32px;"><b>Repetition:</b> Several PCRs were performed to check if the gene fragments were ligated correctly.</li>
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| − | 13_fwd and 13_rev on pT-13-19-15<br/>
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| − | 13_fwd and 19_rev on pT-13-19-15<br/>
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| − | <b>The second one was failed.</b><br/>
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| − | </div>
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| − | <br/>
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| − |
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| − | <b><h1 style="font-size:108%"> Sep.21th</b></h1>
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| − | <div style="padding-left:32px;">1.Medium preparation :BG-11.<br/>
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| − | 2.19_ fwd and 15_rev were used to amplify <i>19-15</i>.
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| − | <br/>
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| − | </div>
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| − | <br/>
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| − | <b><h1 style="font-size:108%"> Sep.22th</b></h1>
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| − | <div style="padding-left:32px;">1.Gel electrophoresis showed that amplification of fragments was successfull.<br/>
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| − | 2.Ligated <i>13</i> and <i>19-15</i> via overlap PCR.<br/>
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| − | 3.This PCR worked well and the products were extracted from the gel using a Agarose Gel DNA Extration Kit.<br/>
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| − | 4.Restriction digest on pCPC-3031-Ni with Sac I.<br/>
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| − | 5.The fragment <i>13-19-15</i> was ligated onto T vector and transformed into E.coli via heat shock.<br/>
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| − | </div>
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| − | <br/>
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| − |
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| − | <b><h1 style="font-size:108%"> Sep.23th</b></h1>
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| − | <div style="padding-left:32px;">1.A colony PCR of pT-13-19-15 was performed with 12 colonies.<br/>
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| − | 2.Two of these colonies containing pT-13-19-15 were used to inoculate overnight cultures.<br/>
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| − | 3.<i>13-19-15</i> gene fragment was phosphorylated.<br/>
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| − | 4.Phosphorylated <i>13-19-15</i> was ligated into pCPC-3031-Ni and transformed into E.coli via heat shock.<br/>
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| − | </div>
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| − | <br/>
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| − | <b><h1 style="font-size:108%"> Sep.24th</b></h1>
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| − | <div style="padding-left:32px;">Plasmids pT-13-19-15 were isolated using a miniprep kit.<br/>
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| − | </div>
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| − | <div class="side-nav">
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| − | <ul>
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| − | <li><a class="topLink" href="#Topp" style="color:#5555FF">Top</a></li>
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| − | <li><a class="topLink" href="#Week1" style="color:#5555FF">Week1</a></li>
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| − | <li><a class="topLink" href="#Week2" style="color:#5555FF">Week2</a></li>
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| − | <li><a class="topLink" href="#Week3" style="color:#5555FF">Week3</a></li>
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| − | <li><a class="topLink" href="#Week4" style="color:#5555FF">Week4</a></li>
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| − | <li><a class="topLink" href="#Week5" style="color:#5555FF">Week5</a></li>
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| − | <li><a class="topLink" href="#Week6" style="color:#5555FF">Week6</a></li>
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| − | <li><a class="topLink" href="#Week7" style="color:#5555FF">Week7</a></li>
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| − | <li><a class="topLink" href="#Week8" style="color:#5555FF">Week8</a></li>
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| − | <li><a class="topLink" href="#Week9" style="color:#5555FF">Week9</a></li>
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| − | <li><a class="topLink" href="#Week10" style="color:#5555FF">Week10</a></li>
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| − | </ul>
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| − | </div>
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| − | <div class="alert alert-info">
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| − | <div class="container-fluid">
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| − | <div class="alert-icon">
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| − | <i class="material-icons">info_outline</i>
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| − | </div>
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| − | <button type="button" class="close" data-dismiss="alert" aria-label="Close">
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| − | <b>Notice:</b> This page is currently under construction. Contents in this page are temporaory and will be modified several times before the final release. — 2016 iGEM Team Tianjin
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