Team:Exeter/lab book/gibs/wk1

From 2012.igem.org

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     <!<p>11.7.12</p>
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     <!<p><b>**Wednesday 11.7.12</b>**</p>
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<p>Dried plasmid DNA of useful parts (chosen parts were a double terminator, TetR promoter, LacI/Ara-1 promoter, pBAD/AraC promoter weak and pBAD/AraC promoter strong) was resuspended in 10 µl milliQ water.</p>
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<p>Dried plasmid DNA of useful parts (chosen parts were a double terminator (BBa_B0014), pBAD weak promoter (BBa_K206001) and pBAD strong promoter (BBa_K206000)) was resuspended according to <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/6" style="color:#1d1d1b"><u>BioBrick extraction</u></a> protocol. Remaining DNA was stored at -20°C</p>
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<p>DNA was left to resuspend for 5 minutes before being put in eppendorf tubes.</p>
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<p>Plasmid DNA was <a href="https://2012.igem.org/Team:Exeter/lab_book/proto/1" style="color:#1d1d1b"><u>transformed into competent cells</u></a></p>
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<p>Tubes were centrifuged briefly (around 5 seconds) to spin down liquid.</p>
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<ul><p>-Tube 1: 50 µl E. coli + 1 µl terminator</p></ul>
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<p>Plasmid DNA was gently pipetted into volumes of competent Top10 E. coli cells like so:</p>
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<ul><p>-Tube 2: 25 µl E. coli + 1 µl pBAD weak</p></ul>
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<p>-Tube 1: 50 µl E. coli + 1 µl terminator</p>
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<ul><p>-Tube 3: 25 µl E. coli + 1 µl pBAD strong</p></ul>
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<p>-Tube 2: 25 µl E. coli + 1 µl TetR</p>
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<p>-Tube 3: 25 µl E. coli + 1 µl LacI/Ara-1 promoter</p>
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<p>-Tube 4: 25 µl E. coli + 1 µl pBad/AraC weak</p>
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<p>-Tube 5: 25 µl E. coli + 1 µl pBad/AraC strong</p>
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<p>Tubes were kept on ice for 30 minutes</p>
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<p>Cells were heat shocked for 30 seconds at 42 *C</p>
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<p>250 µl of S.O.C. medium was added to tube 1</p>
<p>250 µl of S.O.C. medium was added to tube 1</p>
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<p>125 µl of S.O.C. medium was added to tubes 2-5</p>
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<p>125 µl of S.O.C. medium was added to tubes 2 & 3</p>
<p>Tubes were shaken horizontally at 36.8 C, 220rpm for 1 hour</p>
<p>Tubes were shaken horizontally at 36.8 C, 220rpm for 1 hour</p>
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<p>2 LB-agar-antibiotic stocks were made with these ratios:</p>
 
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<p>​1:1000   ampicillin:LB-agar</p>
 
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<p>​1:1000   kanamycin:LB-agar</p>
 
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<p>10 LB(ampicillin) plates were made</p>
 
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<p>2 LB(kanamycin) plates were made</p>
 
<p>Spread plates of 20 and 100 µl volumes of each plasmid transformed E. coli were prepared on LB(ampicillin) plates</p>
<p>Spread plates of 20 and 100 µl volumes of each plasmid transformed E. coli were prepared on LB(ampicillin) plates</p>
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<p>Extra spread plates of 20 and 100 µl volumes of double terminator plasmid were prepared on LB(kanamycin) to check for dual antibiotic resistance.</p>
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<p>Extra spread plates of 20 and 100 µl volumes of double terminator plasmid were prepared on LB(kanamycin) to check for dual antibiotic resistance.</p><br>
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<p>Plates were inverted and incubated overnight at 37 C.</p>
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 <p>12.7.12</p> <p>Chose three colonies from each plate.</p>
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<p>Transferred aseptically into 1:1000 ampicillin:LB broth.</p> <p>Incubated at 37*C, 220rpm overnight.</p>
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<p>13.7.12</p>
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<p>Cultures were transferred to identical sterile falcon tubes and centrifuged at 21 C, 3900 rcf, for 2 minutes.</p>Supernatant was discarded and pellet resuspended in resuspension buffer (250µl)</p>
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<p>250µl lysis solution added</p>
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<p>350µl neutralisation solution added quickly</p>
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<p>Tubes inverted ~3 times to mix.</p>
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<p>Centrifuged at 16100 rcf for 5 minutes, 21 C</p>
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<p>Supernatant added to miniprep column</p>
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<p>Centrifuged at 16100 rcf, 1 min</p>
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<p>Added 500µl wash solution to column</p><p>Centrifuged at 16100 rcf, 1 min. Supernatant discarded.</p>
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<p>Centrifuged again at 16100 rcf, 1 min</p>
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<p>Column switched to new 1.5ml eppendorf</p>
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<p>50µl milli-Q water added</p>
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<p>Incubated at room temperature (21 C) for 2 mins</p>
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<p>Centrifuged for 2 minutes, 16100 rcf.</p>
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<p>Tested DNA concentration with nanodrop spectrophotometer. These concentrations were obtained:</p>
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<p><i>Pbad strong -</i></p>
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<p>1. 130.4</p>
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<p>2. 105.3</p>
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<p>3. 114.2</p>
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<p><i>Tetr -</i></p>
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<p><b>**Thursday 12.7.12**</b></p><br>
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<p>1. 76.8</p>
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<a href="https://2012.igem.org/Team:Exeter/lab_book/proto/2" style="color:#1d1d1b"><u>Colonies were transferred to liquid medium</u></a>
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<p>2. 60.9</p>
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<p>Transferred aseptically into 1:1000 ampicillin:LB broth.</p>
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<p>3. 78.0</p>
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<p>Incubated at 37*C, 220rpm overnight.</p><br>
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<p><i>Plac - </i></p>
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<p><b>**Friday 13.7.12**</b></p><br>
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<p>1. 280.7</p>
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<p>Cultures were transferred to identical sterile falcon tubes and centrifuged at 21 C, 3900 rcf, for 2 minutes.</p>
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<p>2. 272.2</p>
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<p>Miniprep of plasmid DNA according to<a href="http://www.fermentas.com/templates/files/tiny_mce/coa_pdf/coa_k0502.pdf" style="color:#57B947" target="_blank"><u>Fermentas protocol</u></a></p>
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<p>3. 299.2</p>
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<p><b><i>Step 10</i></b>50µl milli-Q water added in place of elution buffer</p>
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<p>Tested DNA concentration with nanodrop spectrophotometer. These concentrations were obtained:</p><br>
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<p><i>pBAD strong -</i></p>
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<p><ul>1. 130.4</ul></p>
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<p><ul>2. 105.3</ul></p>
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<p><ul>3. 114.2</ul></p><br>
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<p><i>Pbad weak -</i></p>
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<p><i>pBAD weak -</i></p>
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<p>1. 186.4</p>
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<p><ul>1. 186.4</ul></p>
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<p>2. 211.4</p>
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<p><ul>2. 211.4</ul></p>
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<p>3. 170.6</p>
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<p><ul>3. 170.6</ul></p><br>
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<p><i>Plasmids digested with following:</i></p>
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<p>Plasmids digested with following:</p><br>
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<p>Plasmid DNA -   4.2 µl</p>
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<p>Plasmid DNA -   4.2 µl</p>
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<p>EcoR1-HF -   0.5 µl</p>
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<p>EcoR1-HF -   0.5 µl</p>
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<p>PstI -   0.5 µl</p>
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<p>PstI -   0.5 µl</p>
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<p>10x NEBuffer 2 -   2.5 µl</p>
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<p>10x NEBuffer 2 -   2.5 µl</p>
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<p>100x BSA -    0.25 µl</p>
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<p>100x BSA -   0.25 µl</p>
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<p>H2O -   7.95 µl</p>
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<p>H2O -   7.95 µl</p><br>
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<p>Incubated for 10 minutes at 37 *C</p>
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<p>Incubated for 10 minutes at 37 °C</p>
<p>DNA run on gel by Becca and Alex to test fragments</p>
<p>DNA run on gel by Becca and Alex to test fragments</p>
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Revision as of 17:11, 25 September 2012

ExiGEM2012 Lab Book Gibs wk1

Operon Construction: 9th - 13th July 2012

 **Wednesday 11.7.12**

Dried plasmid DNA of useful parts (chosen parts were a double terminator (BBa_B0014), pBAD weak promoter (BBa_K206001) and pBAD strong promoter (BBa_K206000)) was resuspended according to BioBrick extraction protocol. Remaining DNA was stored at -20°C

Plasmid DNA was transformed into competent cells

    -Tube 1: 50 µl E. coli + 1 µl terminator

    -Tube 2: 25 µl E. coli + 1 µl pBAD weak

    -Tube 3: 25 µl E. coli + 1 µl pBAD strong

250 µl of S.O.C. medium was added to tube 1

125 µl of S.O.C. medium was added to tubes 2 & 3

Tubes were shaken horizontally at 36.8 C, 220rpm for 1 hour

Spread plates of 20 and 100 µl volumes of each plasmid transformed E. coli were prepared on LB(ampicillin) plates

Extra spread plates of 20 and 100 µl volumes of double terminator plasmid were prepared on LB(kanamycin) to check for dual antibiotic resistance.


**Thursday 12.7.12**


Colonies were transferred to liquid medium

Transferred aseptically into 1:1000 ampicillin:LB broth.

Incubated at 37*C, 220rpm overnight.


**Friday 13.7.12**


Cultures were transferred to identical sterile falcon tubes and centrifuged at 21 C, 3900 rcf, for 2 minutes.

Miniprep of plasmid DNA according toFermentas protocol

Step 1050µl milli-Q water added in place of elution buffer

Tested DNA concentration with nanodrop spectrophotometer. These concentrations were obtained:


pBAD strong -

    1. 130.4

    2. 105.3

    3. 114.2


pBAD weak -

    1. 186.4

    2. 211.4

    3. 170.6


Plasmids digested with following:


Plasmid DNA - 4.2 µl

EcoR1-HF - 0.5 µl

PstI - 0.5 µl

10x NEBuffer 2 - 2.5 µl

100x BSA - 0.25 µl

H2O - 7.95 µl


Incubated for 10 minutes at 37 °C

DNA run on gel by Becca and Alex to test fragments

Retrieved from "http://2012.igem.org/Team:Exeter/lab_book/gibs/wk1"