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| Line 611: |
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| | <p>Determination of the medium that will be use => LB or PBS1X</p> | | <p>Determination of the medium that will be use => LB or PBS1X</p> |
| | </div> | | </div> |
| − | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo42">July 27</button> | + | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo42">August 27</button> |
| | <div id="demo43" class="collapse"> | | <div id="demo43" class="collapse"> |
| − | <p>Colony PCR and electrophoresis of “08”, “15”, “12-02” and “30-02” to check the size of the insert. Positive results for “15”, “12-02” and “13-02” (expected size) but negative result for “08” (several sizes observed). Starters of “08”, “15”, “12-02” and “30-02”.</p> | + | <p>Ligation K823013 + I13504 + pSB1C3</p> |
| − | <p>Plasmid purification (miniprep) of “05”, “09” and “10”. Problem encountered: genomic DNA contamination.</p> | + | |
| − | <p>E/P digestion and electrophoresis to check the size of the insert of “05”, “09”, and “10”. </p> | + | <p>Transformation of K823013 + I13504 +pSB1K3 into <i>E.coli</i> (DH5-Alpha)</p> |
| | + | |
| | + | <p>Test on many machines to observe GFP => confocal microscope, magic-box, TECAN, and fluorimeter</p> |
| | </div> | | </div> |
| − | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo44">July 28</button> | + | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo44">August 28</button> |
| | <div id="demo44" class="collapse"> | | <div id="demo44" class="collapse"> |
| − | <p>Miniprep of “08”, “15”, 12-02”, “30-02”. E/P digestion and electrophoresis to check the size of the insert of “08”, “15”, 12-02”, “30-02”. Positive results for “15”, “12-02”, “30-02” (expected size) and negative result for “08” (unexpected size). </p> | + | <p>Transformation of K823013 + I13504 +pSB1C3 gives no transformants</p> |
| − | <p>Comment: constructs “12-02” and “30-02” have to be compared with the single “12” and “30” to check if “02” was added. </p>
| + | |
| | + | <p>Measure of GFP intensity with TECAN and confocal microscope on “18-17”, “19-17”, “20-17”, “21-17”, “22-17”, “23-17”, “01-24”, “33” and “32”</p> |
| | </div> | | </div> |
| − | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo45">July 29</button> | + | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo45">August 31</button> |
| | <div id="demo46" class="collapse"> | | <div id="demo46" class="collapse"> |
| − | <p>Colony PCR and electrophoresis to check the size of the insert of “08”. Negative result</p>
| + | Digestion E/P of K823013 + I13504 + pSB1C3 |
| − | <p>Electrophoresis of “12”, “12-02”, “30” and “30-02”. No result (no DNA observed on the agarose gel)</p>
| + | |
| − | <p>Construction of “09-02”, “10-02” and “13-02”: E/S digestion of “09”, “10” and “13”. X/P digestion of “02”. Ligation into pSB1A3, transformation into TG1. Positive results for “09-02” and “13-02” (1 colony on the plate). Negative result for “10-02” (no colony on the plate).</p>
| + | |
| − | <p>Resuspension of biobrick “11” and new try for the “05” and the “08” received from IDT. E/P digestion and ligation into pSB1C3. Transformation into TG1. Positive result for “11” (several colonies on the plate) but not for “05” and “08” (no colony on the plate) </p>
| + | |
| | </div> | | </div> |
| − | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo46">July 30</button> | + | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo46">September 3</button> |
| | <div id="demo46" class="collapse"> | | <div id="demo46" class="collapse"> |
| − | <p>Colony PCR and electrophoresis to check the size of the insert of « 11 », “09-02” and “13-02”. Uncertain results (problems: DNA is not observed on the agarose gel). “13-02” seems good: starter of “13-02”.</p> | + | <p>Measure of GFP intensity with fluorimeter and magic box on “18-17”, “19-17”, “20-17”, “21-17”, “22-17”, “23-17”, “01-24”, “33” and “32”</p> |
| − | <p>New try with new oligos for the colony PCR and electrophoresis of « 08 ». No result (no DNA observed on the agarose gel).</p>
| + | |
| − | <p>New try with new volumes for resuspension of the biobrick “05” received from IDT. E/P digestion and ligation into pSB1C3. Transformation into TG1. Negative result (no colony on the plate).</p>
| + | <p>Sequencing result: “23-17” is bad </p> |
| − | <p>New try to construct “09-02”, “10-02” and “13-02”. E/S digestion of “09”, “10” and “13”. X/P digestion of “02”. Ligation overnight into pSB1A3, transformation into TG1 (07/31). Positive result for “13-02” (several colonies on the plate. Negative result for “09-02” and “10-02” (no colony on the plate).</p>
| + | |
| − | <p>E/P digestion to check the size of the insert of “30”, “30-02”, “12”, “12-02”. Results seems good for “12” and “12-02”: send for sequencing. Negative results for “30”, “30-02” (unexpected sizes).</p> | + | |
| | </div> | | </div> |
| − | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo47">July 31</button> | + | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo47">September 4</button> |
| | <div id="demo47" class="collapse"> | | <div id="demo47" class="collapse"> |
| − | <p>Miniprep of « 13-02 ». E/P digestion and electrophoresis to check the size of the insert. Positive result (expected size).</p>
| + | Preparation of standard curve of Atton 488 for magic-box, fluorimeter and TECAN |
| − | <p>Colony PCR and electrophoresis to check the size of the insert of “13-02”. Negative result (unexpected size).</p>
| + | |
| − | <p>Transformation of “09-02”, “10-02” and “13-02” into TG1.</p>
| + | |
| − | <p>pSB1C3 backbone was running out. New backbone done: E/P digestion on measurement biobricks, electrophoresis and purification of the digested plasmid by gel extraction and PCR clean up. </p><p>Problem encountered: no DNA observed on the agarose gel. </p>
| + | |
| | </div> | | </div> |
| | </div> | | </div> |
| | | | |
| | | | |
| − | <div id="main" style="position:relative;top:0px;left:172px;width:1000px;" class="menu-container">
| |
| − | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo48">August 3</button>
| |
| − | <div id="demo48" class="collapse">
| |
| − | <p>Colony PCR and electrophoresis to check the size of the insert of “09-02”, “10-02” and “13-02”. Positive results (expected sizes). Starters of “09-02”, “10-02” and “13-02.</p>
| |
| − | <p>New try for making pSB1C3 backbone: E/P digestion of measurement biobricks, electrophoresis and purification of the digested plasmid by gel extraction and PCR clean up. </p>
| |
| − | <p>Resuspension of the biobrick “07” (cotA) received from IDT. E/P digestion and ligation into pSB1A3, transformation into TG1</p>
| |
| − | </div>
| |
| − | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo49">August 4</button>
| |
| − | <div id="demo49" class="collapse">
| |
| − | <p>Colony PCR and electrophoresis to check the size of the insert on “07” => we got a positive clone, starter of this clone launched</p>
| |
| − | <p>Plasmid purification of “13-02” “09-02” “10-02”</p>
| |
| − | <p>E/P digestion of “05” and “11” and ligation “05” + pSB1K3 and “11” + pSB1K3 </p>
| |
| − | <p>Transformation of ligation products into E.coli (TG1)</p>
| |
| − | <p>PCR on “07” and “08” => seems to be unclean with the Q5 high fidelity DNA amplification</p>
| |
| − | <p>X/P digestion of “15” </p>
| |
| − | <p>Overnight ligation “12”+”02”+pSB1A3 and “01”+”15”+pSB1T3</p>
| |
| − | <p>“30-02” and “08” got bad results in sequencing</p>
| |
| − | </div>
| |
| − | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo50">August 5</button>
| |
| − | <div id="demo50" class="collapse">
| |
| − | <p>Transformation of ”01-15” and “12-02” into E.coli (TG1)</p>
| |
| − | <p>Plasmid purification of “09-02” “07” “13-02” “09-02” “10-02” => low concentration of “09-02” but we got good result for “13-02”</p>
| |
| − | <p>Transformation of “12-02” and “01-15” into TG1</p>
| |
| − | <p>PCR clean up “07” “08” “24” ==> low plasmid concentration of “07” and “08” to do again. </p>
| |
| − | </div>
| |
| − | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo51">August 6</button>
| |
| − | <div id="demo51" class="collapse">
| |
| − | <p>Colony PCR of “05” “11” “12-02” “01-15” => good but only for “12-02”</p>
| |
| | | | |
| − | <p>PCR clean up of “08” and “07” → PCR products contaminated → PCR clean up</p>
| |
| − |
| |
| − | <p>Transformation of “10-02” and “09-02”→ Wrong size starter relaunched</p>
| |
| − |
| |
| − | <p>Ligation “09-02” and “10-02” into pSB1A3 and “01-15” into pSB1T3</p>
| |
| − | </div>
| |
| − | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo52">August 7</button>
| |
| − | <div id="demo52" class="collapse">
| |
| − | <p>Colony PCR of “05” and “01-15” --> no results on this PCR </p>
| |
| − |
| |
| − | <p>Transformation of “09-02” “10-02” “01-15” into E.coli (TG1)</p>
| |
| − |
| |
| − | <p>Ligation “05” + pSB1K3 and “11” + pSB1K3</p>
| |
| − |
| |
| − | <p>Plasmid purification of “12-02” --> Bad results with electrophoresis</p>
| |
| − |
| |
| − | <p>For “05” “07” “08” “11” → PCR with Q5 high fidelity polymerase and “PCR clean up”</p>
| |
| − |
| |
| − | <p>Preparation of BL21 competent cells </p>
| |
| − | </div>
| |
| − | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo53">August 10</button>
| |
| − | <div id="demo53" class="collapse">
| |
| − | <p>Colony PCR of “01-15” → No results => Ligation with the digestion product “01-15” relaunched</p>
| |
| − |
| |
| − | <p>For “05” “07” “08” “11” : Digestion E/S + Ligation pSB1C3 + Transformation into TG1</p>
| |
| − |
| |
| − | <p>For “30” : Digestion E/P + Ligation into pSB1K3</p>
| |
| − |
| |
| − | <p>For “01-15” : Ligation into pSB1T3</p>
| |
| − |
| |
| − | <p>PCR clean up of “28” </p>
| |
| − | </div>
| |
| − | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo54">August 11</button>
| |
| − | <div id="demo54" class="collapse">
| |
| − | <p>Colony PCR of “09-02” “10-02” “05” “07” “08” “11”</p>
| |
| − |
| |
| − | <p>Preparation of TG1 competent cells </p>
| |
| − |
| |
| − | <p>We used the wrong “02” Biobrick we have to do everything again with the good one → Colony PCR of the “new” “02” → size = ok </p>
| |
| − |
| |
| − | <p>Transformation of “30” “01-01” “02” into TG1 </p>
| |
| − | </div>
| |
| − | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo55">August 12</button>
| |
| − | <div id="demo55" class="collapse">
| |
| − | <p>Preparation of the backbone pSB1A3</p>
| |
| − |
| |
| − | <p>Ligation of “05” “08” “07” “11” into pSB1A3</p>
| |
| − |
| |
| − | <p>Colony PCR of “30” “21-17” “23-17” “01-15”</p>
| |
| − | <p>For “35” et “36” : Digestion E/S + ligation into pSB1A3 + transformation into TG1</p>
| |
| − | <p>For “37” “38” : Digestion E/P + Ligation into pSB1K3 + transformation into “supercompetent” cells</p>
| |
| − |
| |
| − | <p>Plasmid purification of “02” --> Low concentration of plasmid, starter relaunched</p>
| |
| − |
| |
| − | <p>For “02” : Digestion X/P </p>
| |
| − | <p>For “13” : Digestion E/S</p>
| |
| − |
| |
| − | <p>Ligation of “35-02” “36-02” “12-02” “13-02” into pSB1A3</p>
| |
| − |
| |
| − | <p>PCR on “09” et “10” to remove the stop codon</p>
| |
| − |
| |
| − | <p>Transformation of “05” “07” “08” “11” into “supercompetent” cells </p>
| |
| − | </div>
| |
| − | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo56">August 13</button>
| |
| − | <div id="demo56" class="collapse">
| |
| − | <p>Colony PCR on “05” “07” “08” “21-17” “11” “35” “36” “37” “38”</p>
| |
| − |
| |
| − | <p>Transformation of “12-02” “13-02” “35-02” “36-02” “36-02” into TG1</p>
| |
| − |
| |
| − | <p>PCR clean up of “35” “36” </p>
| |
| − |
| |
| − | <p>Digestion E/S of “12” “35” “36” “16” “13”</p>
| |
| − |
| |
| − | <p>For “12-02” “13-02” “35-02” “36-02” : Ligation into pSB1A3 + transformation in TG1</p>
| |
| − | <p>For “01-15” : Ligation into pSB1T3 + transformation into TG1</p>
| |
| − | <p>For “16-17” : Ligation into pSB1C3 + transformation into TG1</p>
| |
| − |
| |
| − | <p>Colony PCR on “38” “05” “07” “36” ”08” “11”</p>
| |
| − |
| |
| − | <p>For “39” “30” : Digestion E/P + Ligation into pSB1K3</p>
| |
| − | </div>
| |
| − | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo57">August 14</button>
| |
| − | <div id="demo57" class="collapse">
| |
| − | <p>Colony PCR on “12-02” “13-02” “39” “36-02” “23-17” “35-02”</p>
| |
| − |
| |
| − | <p>Plasmid purification of “36” “38” “07” “05” “35” “11” “37” --> Verification ok for “36” “38” “35” “37” </p>
| |
| − | </div>
| |
| − | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo58">August 15</button>
| |
| − | <div id="demo58" class="collapse">
| |
| − | <p>Colony PCR on “05” “07” “08” “11” “16-17” “21-17” “05” “07” “08” “11” “16-17” “21-17” --> Bad results for all</p>
| |
| − |
| |
| − | <p>Plasmid purification of “38” “05” “11” “08” “39” “12-02” “13-02” “35-02” “23-17” “35” “01-15” “16-17” “21-17” --> Problem with the ladder, we cannot read the gel</p>
| |
| − | </div>
| |
| − | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo59">August 17</button>
| |
| − | <div id="demo59" class="collapse">
| |
| − | <p>For “38” “39” “08” “05” : Digestion E/S</p>
| |
| − | <p>For “12” “13” “39” “35” “35-02” “13-02” “12-02” : Digestion X/P</p>
| |
| − |
| |
| − | <p>For “38-12” “38-13” “39-02” “38-39” “08-02” “36” “05-02”: Ligation into pSB1A3</p>
| |
| − | <p>For “01-3502” “01-1302” “01-1202” “01-35” : Ligation into pSB1T3</p>
| |
| − | <p>For “21-17” “22-17” : Ligation into pSB1C3</p>
| |
| − | ==> Transformation for all ligation products into TG1
| |
| − |
| |
| − | <p>Colony PCR of “07” “05” “08” “11” with “pool” technic</p>
| |
| − | </div>
| |
| − | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo60">August 18</button>
| |
| − | <div id="demo60" class="collapse">
| |
| − | <p>Colony PCR of “11” “7” => starters launched on positive clones</p>
| |
| − |
| |
| − | <p>Plasmid verification of “05” “07” “08” “11” and “16-17”</p>
| |
| − | <p>“07” and “08” have to be relaunched </p>
| |
| − |
| |
| − | <p>Colony PCR on transformants from August 17 => Starters on positive clones launched and the constructions “01-3502” and “38-39” have to be rebuilt</p>
| |
| − |
| |
| − | <p>Preparation of the backbone pSB1A3</p>
| |
| − |
| |
| − | <p>Ligation of “01-1302” “01-1202” “01-35” into pSB1T3</p>
| |
| − | </div>
| |
| − | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo61">August 19</button>
| |
| − | <div id="demo61" class="collapse">
| |
| − | <p>Starters launched of “05” and “11”</p>
| |
| − | <p>Preparation of the backbone pSB1A3</p>
| |
| − | <p>Sequencing results “12-02” “13-02” “35-02” “01-15” are ok</p>
| |
| − | <p>“08” is bad and “05” couldn’t be sequenced</p>
| |
| − | <p>Plasmid purification of “30” “38-13” “39-02” “08-02” “05-02” “38-12” “11” “07” “16-17”</p>
| |
| − | <p>“38-13” “39-02” “16-17” “38-12” send for sequencing</p>
| |
| − | <p>“30” “08-02” “05-02” “11” “07” are bad we cannot use its </p>
| |
| − | </div>
| |
| − | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo62">August 20</button>
| |
| − | <div id="demo62" class="collapse">
| |
| − | <p>Colony PCR of “05-02” “01-1302”</p>
| |
| − |
| |
| − | <p>Plasmid purification of “05” and “11” and of 2 starters to make some backbones (pSB1A3 and pSB1T3)</p>
| |
| − | <p>The digestion to prepare pSB1T3 is bad, starter relaunched</p>
| |
| − |
| |
| − | <p>Ligation of “38-39” “11-02” into pSB1A3</p>
| |
| − | <p>“38-11” into pSB1C3</p>
| |
| − | <p>“01-1202” “05-02” “01-36” “01-1302” “01-3502” “01-35” “21-17” “22-17” into pSB1K3</p>
| |
| − | <p>Transformation of previous ligation products into DH5-Alpha for “21-17” and “22-17”, into TG1 for “11-02” “38-11” “05-02” and “38-39” and into BL21 for “01-1202” “01-36” “01-1302” “01-3502” and “01-35”</p>
| |
| − |
| |
| − | <p>“38-13” “39-02” “11” and “38-12” sent for sequencing</p>
| |
| − | <p>Sequencing result of “05” => bad </p>
| |
| − |
| |
| − | <p>Transformation of “18-17” “19-17” “20-17” and “23-17” into DH5-Alpha</p>
| |
| − |
| |
| − | <p>Colony PCR of “07”</p>
| |
| − | </div>
| |
| − | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo63">August 21</button>
| |
| − | <div id="demo63" class="collapse">
| |
| − | <p>Digestion E/S fof “16” “38” “11” “22”, X/P for “17” “39” “02” “11” “35”</p>
| |
| − |
| |
| − | <p>Ligation of “16-17” “22-17” + pSB1C3 “38-39” “11-02” “38-11” + pSB1A3</p>
| |
| − | <p>“01-35” “05” “07” “08” “30” + pSB1K3</p>
| |
| − |
| |
| − | <p>Transformation of “30” “08” “07” “05” into C2987 (“supercompetent” cells)</p>
| |
| − |
| |
| − | <p>Transformation of “38-39” “11-02” “38-11” and “01-35” into TG1</p>
| |
| − |
| |
| − | <p>Plasmid purification of “01-1202” “01-3502” “05” “01-1302” “07” and of pSBT3 to get some backbone.</p>
| |
| − |
| |
| − | <p>Colony PCR of “08” “01-3502” “01-1202” “01-1302” “01-36”</p>
| |
| − |
| |
| − | <p>We got many bad results today so bad luck, but we got some good results also.</p>
| |
| − | </div>
| |
| − | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo64">August 22</button>
| |
| − | <div id="demo64" class="collapse">
| |
| − | <p>Colony PCR of “38-39” “38-11” “11-02” “01-35” </p>
| |
| − | <p>Starters launched of positive clones</p>
| |
| − |
| |
| − | <p>Transformation of overnight ligation produtcs (“16-17” and “22-17” from August 21)</p>
| |
| − |
| |
| − | <p>Plasmid purification to make some pSB1A3 and pSB1T3</p>
| |
| − |
| |
| − | <p>First protein expression test, we maybe succeed to express our first laccase </p>
| |
| − | </div>
| |
| − | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo65">August 23</button>
| |
| − | <div id="demo65" class="collapse">
| |
| − | <p>Colony PCR of “16-17” and “22-17” launched overnight => green colonies!!</p>
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| − |
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| − | <p>Starters launched of positive results</p>
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| − |
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| − | <p>Plasmid purification of “22”-17” “16-17” and ‘01-35” => all results are good!!!!</p>
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| − |
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| − | <p>Digestion E/S for “38” and X/P for “39”, ligation overnight into pSB1A3</p>
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| − | </div>
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| − | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo66">August 24</button>
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| − | <div id="demo66" class="collapse">
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| − | <p>Transformation of “30” “08” “07” “11” “05” into TG1</p>
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| − |
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| − | <p>Plasmid purification of “16-17” “01-1302” “01-1202” “22-17” “01-3502”</p>
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| − |
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| − | <p>Colony PCR of “05” “07” “08” “11” => no positive results!</p>
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| − |
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| − | <p>Q5 PCR of “05” “07” “08” “11” “30” => positive results </p>
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| − |
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| − | <p>“05” and “01-35” sent for sequencing </p>
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| − | </div>
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| − | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo67">August 25</button>
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| − | <div id="demo67" class="collapse">
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| − | <p>Transformation of “05” “07” “08” “11” “30” into TG1 </p>
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| − |
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| − | <p>Plasmid purification of “01-36” “19-17” “18-17” “23-17” “20-17” => good results</p>
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| − |
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| − | <p>“01-1202” “01-1302” “01-3502” sent for sequencing</p>
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| − |
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| − | <p>Sequencing results of “01-35” “38-12” “38-13” “39-02” => good results “05” “11” => bad results</p>
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| − |
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| − | <p>Starters launched of “39” “01-1302” “01-1202” “01-3502” “01-15” “13-02” “12-02” “38”</p>
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| − | </div>
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| − | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo68">August 26</button>
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| − | <div id="demo68" class="collapse">
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| − | Protein expression => the cytochrome c and the laccase are expressed.
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| − | </div>
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| − | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo69">August 27</button>
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| − | <div id="demo69" class="collapse">
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| − |
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| − | <p>Isolation and starter launched of “32”</p>
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| − |
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| − | <p>Plasmid purification of “39” “01-1302” “01-1202” “01-3502” “01-15” “13-02” “12-02” “38” “16-17”=> no results!</p>
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| − |
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| − | <p>Ligation of “40” “41” “42” “43” “44” “45” “46” “47” “48” “22-17” + pSB1C3</p>
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| − | <p>“30” + pSB1K3</p>
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| − |
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| − | <p>Transformation of “30” “40” “45” “48” into TG1</p>
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| − | <p>Transformations didn’t work! => “40” “45” “48” relaunched </p>
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| − |
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| − | <p>Sequencing results “01-1302” => without promoter</p>
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| − | <p>“01-1202” and “01-3502” are good</p>
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| − |
| |
| − | </div>
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| − | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo70">August 31</button>
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| − | <div id="demo70" class="collapse">
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| − | <p>Colony PCR of “40” “45” “48” </p>
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| − |
| |
| − | <p>Starters launched of “18-17” “19-17” “20-17” “21-17” “22-17” “23-17” “32” “33” “01-24” each in triplicate</p>
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| − |
| |
| − | <p>Stock of backbone pSB1C3 done</p>
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| − |
| |
| − | <p>Preparation of SDS-PAGE, of solutions and of the column to make the protein purification</p>
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| − |
| |
| − | <p>Digestion E/P of “22-17” “43” “44” “47” and digestion X/P of “1302” “3902”</p>
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| − |
| |
| − | <p>Ligation “43” “44” “47” into pSB1C3</p>
| |
| − | <p>“013002” into pSB1K3</p>
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| − | <p>“01-3902” “01-1302” into pSB1C3 </p>
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| − | </div>
| |
| − | </div>
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| − |
| |
| − | <div id="main" style="position:relative;top:0px;left:172px;width:1000px;" class="menu-container">
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| − | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo71">September 1</button>
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| − | <div id="demo71" class="collapse">
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| − | <p>Colony PCR of “40” “45” “48” all clones are positive, starters launched</p>
| |
| − |
| |
| − | <p>Protein purification of “01-3502” “01-1202” </p>
| |
| − | <p>SDS-PAGE of “01-3502” “01-1202”</p>
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| − |
| |
| − | <p>01-3502 purified and pure!!!! We got our first protein which is a laccase from E.coli</p>
| |
| − |
| |
| − | <p>Starters launched of “38” “13-02” “01-36” “01-15” “01-3502” “01-1202” “37” “35-02” “12-02” “35” “36” “01-35” “39” “40” “45” “48” </p>
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| − | </div>
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| − |
| |
| − | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo72">September 2</button>
| |
| − | <div id="demo72" class="collapse">
| |
| − | <p>Plasmid purification of “01-36” “40” “39” “48” “45” “37” “36” “01-1202” “01-36” “01-15” “35-02” “35” “38” “13-02” “12-02” “01-3502” “01-35”</p>
| |
| − |
| |
| − | <p>Protein expression of 01-3502 and 01-1202</p>
| |
| − |
| |
| − | <p>Colony PCR of “01-15” beause we have doubt concerning colonies => all colonies were positive</p>
| |
| − |
| |
| − | <p>Ligation of “43” “44” “47” “03002” “01-1302” “013902” into pSB1C3</p>
| |
| − | <p>Transformation of “43” “44” “47” “013002” into C2987 and “01-1302” “01-3902” “3813-02” “3812-02” into TG1</p>
| |
| − | </div>
| |
| − | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo73">September 3</button>
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| − | <div id="demo73" class="collapse">
| |
| − | <p>Verification digestion on “40” “38” “01-15” “48” “45” “01-36” “01-1202” “01-36” “37” </p>
| |
| − |
| |
| − | <p>Plasmid purification of “01-1202” </p>
| |
| − |
| |
| − | <p>Colony PCR of “3812-02” “3813-02” “01-3902” “01-1302” starters launched on positive results</p>
| |
| − | <p>No colony for “43” “44” “47” “013002”</p>
| |
| − |
| |
| − | <p>Preparation of the backbone pSB1C3</p>
| |
| − |
| |
| − | <p>Transformation of “013002” “41” “44” “47” into C2987</p>
| |
| − | </div>
| |
| − | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo74">September 4</button>
| |
| − | <div id="demo74" class="collapse">
| |
| − | <p>Plasmid purification of “45” “01-36” “381302” “38” “01-1202” “3812-02” “3813-02”</p>
| |
| − |
| |
| − | <p>Colony PCR of “47” “01-3002” “43” “44” => almost everything is positive!!! Great day!</p>
| |
| − | </div>
| |
| − |
| |
| − | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo75">September 7</button>
| |
| − |
| |
| − | <div id="demo75" class="collapse">
| |
| − | <p>Plasmid purification (miniprep) of “44” “47” “43” “38” “01-1302” “01-1202” “01-3002” “45” </p>
| |
| − |
| |
| − | <p>“01-1302” “01-36” “3813-02” seems good so they are send for sequencing.</p>
| |
| − |
| |
| − | <p>Construction : Digestion E/S of “38” “01-36” “01-35” “01” and Digestion X/P of “44” “47” “43” “48” “44” “45” “1302” “3813-02” “39-02” “40” “3812-02”</p>
| |
| − |
| |
| − | <p>Starter launched on “01-1202” “01-3002” for production and on “0136” for miniprep</p>
| |
| − |
| |
| − | </div>
| |
| − | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo76">September 8</button>
| |
| − | <div id="demo76" class="collapse">
| |
| − | <p>Ligation in pSB1C3 of “0136-02” “01-381302” “01-381202” “01-3902” “01-1302” “38-3902” “0136-40” “0136-45” “0136-48” “0135-43” “0135-44” “0135-47”</>
| |
| − |
| |
| − | <p>Transformation of “01-3002” in BL21, of pSB4K5 and pSB3T5 in TG1 and transformation of ligation product in TG1</p>
| |
| − |
| |
| − | <p>Purification of “01-1202” (1) and (2):</p>
| |
| − | <p>Induction by IPTG </p>
| |
| − | <p>Storing the cells at -80°C </p>
| |
| − | </div>
| |
| − |
| |
| − | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo77">September 9</button>
| |
| − | <div id="demo77" class="collapse">
| |
| − | <p>Colony PCR of “013002” “013002” => all results are positive</p>
| |
| − |
| |
| − | <p>Production of “01-1202” : Sonication of cells, centrifugation 10 min at 4500 rpm, ultracentrifugation 45 min at 4500 rpm, solubilizing membrane in Triton 1% during 2h and incubation overnight.</p>
| |
| − | </div>
| |
| − | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo78">September 10</button>
| |
| − | <div id="demo78" class="collapse">
| |
| − | <p>Preparation of SDS Page 15% and a western blot with products of yesterday => results are not good so we have to resart the production</p>
| |
| − | </div>
| |
| − | <button type="button" class="btn btn-info" data-toggle="collapse" style="width: 110px;" data-target="#demo79">September 14</button>
| |
| − | <div id="demo79" class="collapse">
| |
| − | <p>Culture of “01-1202” and “01-3002” in anaerobic condition, cells were centrifuged 10 min at 4500 rpm and pellet were stored at -20°C</p>
| |
| − | <p>A western blot was done → Production of Cytochrome, monomer in the membrane and dimer in the cytoplasm</p>
| |
| − |
| |
| − | <p>The next step is to purify “01-1202” and “01-3002” and start a culture on 01-3502 in anaerobic condition</p>
| |
| − | </div>
| |
| − | </div>
| |
| | | | |
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