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Team:Exeter/lab book/gibs/wk2
From 2012.igem.org
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| - | <!<p>16.7.12</p> | + | <!<p><b>16.7.12</b></p> |
<p>Protocol as on 11.7.12 (basic transformation procedure.)</p> | <p>Protocol as on 11.7.12 (basic transformation procedure.)</p> | ||
<p>TetrRBS and RBS bio brick sequences resuspended in 10µl water</p> | <p>TetrRBS and RBS bio brick sequences resuspended in 10µl water</p> | ||
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<p>Spread plates of 20µl and 100µl transformed E. coli made.</p> | <p>Spread plates of 20µl and 100µl transformed E. coli made.</p> | ||
<p>Overnight incubation at 37 °C</p> | <p>Overnight incubation at 37 °C</p> | ||
| - | + | <p></p> | |
| - | <p>17.7.12</p> | + | <p><b>17.7.12</b></p> |
<p>Made up LB-agar for later use</p> | <p>Made up LB-agar for later use</p> | ||
<p>Chose 3 colonies from each 100 µl spread plate.</p> | <p>Chose 3 colonies from each 100 µl spread plate.</p> | ||
<p>Colonies transferred by pipette tip into 5 ml LB(amp) broth (1:1000 ratio)</p> | <p>Colonies transferred by pipette tip into 5 ml LB(amp) broth (1:1000 ratio)</p> | ||
<p>Incubated at 37 °C, 220 rpm, overnight</p> | <p>Incubated at 37 °C, 220 rpm, overnight</p> | ||
| - | + | <p></p> | |
| - | <p>18.7.12 (miniprep attempt)</p> | + | <p><b>18.7.12 (miniprep attempt)</b></p> |
<p>Centrifuged tubes, 4℃, 10 minutes, 3901 rcf</p> | <p>Centrifuged tubes, 4℃, 10 minutes, 3901 rcf</p> | ||
<p>Pellet was considered too small, probably due to shaking incubator turning off</p> | <p>Pellet was considered too small, probably due to shaking incubator turning off</p> | ||
<p>4 colonies were selected from plates, put in broth and then incubated (as in 17.7.12)</p> | <p>4 colonies were selected from plates, put in broth and then incubated (as in 17.7.12)</p> | ||
| - | + | <p></p> | |
| - | <p>19.7.12 (miniprepping)</p> | + | <p><b>19.7.12 (miniprepping)</b></p> |
<p>Transferred cultures to identical falcon tubes to remove pipette tips</p> | <p>Transferred cultures to identical falcon tubes to remove pipette tips</p> | ||
<p>Centrifuged at 3901 rcf for 5 minutes, 4 °C</p> | <p>Centrifuged at 3901 rcf for 5 minutes, 4 °C</p> | ||
Revision as of 19:25, 28 July 2012
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Operon Construction: 16th - 20th July 2012 16.7.12Protocol as on 11.7.12 (basic transformation procedure.) TetrRBS and RBS bio brick sequences resuspended in 10µl water Resuspended DNA (1µl) added to top10 competent cells (25µl) Incubated on ice for 30 minutes 42 °C heat shock for 30 seconds Incubated on ice for 2 minutes S.O.C. Medium aseptically added (250µl) Incubated in shaking incubator - 37 C, 220 rpm, 1 hour LB(amp) (1:1000) plates made Spread plates of 20µl and 100µl transformed E. coli made. Overnight incubation at 37 °C 17.7.12 Made up LB-agar for later use Chose 3 colonies from each 100 µl spread plate. Colonies transferred by pipette tip into 5 ml LB(amp) broth (1:1000 ratio) Incubated at 37 °C, 220 rpm, overnight 18.7.12 (miniprep attempt) Centrifuged tubes, 4℃, 10 minutes, 3901 rcf Pellet was considered too small, probably due to shaking incubator turning off 4 colonies were selected from plates, put in broth and then incubated (as in 17.7.12) 19.7.12 (miniprepping) Transferred cultures to identical falcon tubes to remove pipette tips Centrifuged at 3901 rcf for 5 minutes, 4 °C Supernatant tipped away 250µl resuspension solution added Pellet resuspended by gentle pipetting up and down 250 µl lysis solution added Tubes inverted 350 µl neutralisation solution rapidly added Tubes inverted Centrifuged at 13000rpm, 5 minutes, 4 °C Supernatant transferred to geneJET miniprep column Centrifuged at 13000rpm, 1 minute Flow through discarded 500 µl wash solution added Centrifuged at 13000rpm, 30 seconds Discard flow through 500 µl wash solution added Centrifuged at 13000rpm, 1 minute Discard flow through Centrifuged at 13000rpm, 1 minute Transferred column to a new 1.5 ml eppendorf 50 µl elution buffer added Incubated for 2 minutes, room temperature |
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