Team:Amsterdam/extra/protocols

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<h1>Protocols</h1><br/>
<h1>Protocols</h1><br/>
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<center>__TOC__</center>
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__NOTOC__
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<div class='clear'></div>
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</div>
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<div id="sub-menu" class="content-block">
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<h2>Transformation Protocol in DH5a (Invitrogen)</h2>
<h2>Transformation Protocol in DH5a (Invitrogen)</h2>
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*50 ºC ----- 1 hour<br/>
*50 ºC ----- 1 hour<br/>
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[[File:Amsterdam_Gibson_pic.png|700px]]<br\>
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Adapted from:<br\>
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http://www.syntheticgenomics.com <br\>
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http://eu.idtdna.com
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Notes:
Notes:
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'''Growth curve time points'''<br\>
'''Growth curve time points'''<br\>
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The assessment Jose suggested seems fine.
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This contained a time laps of 30min for the first period of time (lets say first 3 hours) and after that time lapses of a hour would be better.
This contained a time laps of 30min for the first period of time (lets say first 3 hours) and after that time lapses of a hour would be better.
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*Prepare 2x 10ml pSB1AT3 + LacH cultures (in normal LB and with appropriate antibiotic added)
*Prepare 2x 10ml pSB1AT3 + LacH cultures (in normal LB and with appropriate antibiotic added)
*Prepare 2x 10ml pSB1AT3 + pBAD cultures (in normal LB and with appropriate antibiotic added)
*Prepare 2x 10ml pSB1AT3 + pBAD cultures (in normal LB and with appropriate antibiotic added)
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*Incubate at approximately 37 C for 20 hours (starting time 21:20)
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*Incubate at approximately 37 C for 20 hours  
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'''Day 2 (14-9-2012)'''<br\>
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'''Day 2 '''<br\>
Setup:
Setup:
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*Stop incubation of cultures after 20 hours of growth (at 16:20), cultures should now be well into the stationary faze.
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*Stop incubation of cultures after 20 hours of growth , cultures should now be well into the stationary faze.
*Prepare 8x a 500ml flasks containing 49.5ml of LB each
*Prepare 8x a 500ml flasks containing 49.5ml of LB each
*For 4 flasks add appropriate antibiotic for culture 1 (pSB1AT3 + LacH containing bacteria from day 1 - step 1)
*For 4 flasks add appropriate antibiotic for culture 1 (pSB1AT3 + LacH containing bacteria from day 1 - step 1)
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*To the 4 flasks from step 3 add 0.5ml of culture 1
*To the 4 flasks from step 3 add 0.5ml of culture 1
*To the 4 flasks from step 4 add 0.5ml of culture 2
*To the 4 flasks from step 4 add 0.5ml of culture 2
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*Incubate new cultures (step 5 & 6) at 37 degrees for 18 hours (starting time 18:00)
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*Incubate new cultures (step 5 & 6) at 37 degrees for 18 hours  
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'''Day 3 (15-9-2012)'''<br\>
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'''Day 3 '''<br\>
Experiment:
Experiment:
*Stop incubation of cultures after 18 hours of growth (at 12:00), cultures should now be well into the stationary faze.
*Stop incubation of cultures after 18 hours of growth (at 12:00), cultures should now be well into the stationary faze.
*From all 8 cultures take 1.5 ml sample and put on ice (-20 C)  these are negative control reference points (IPTG- and ARA-)
*From all 8 cultures take 1.5 ml sample and put on ice (-20 C)  these are negative control reference points (IPTG- and ARA-)
*To 3 of 4 cultures) (day 2 – step 5) each add 100mM IPTG, these will become the IPTGex30, IPTGex60, IPTGex120 cultures
*To 3 of 4 cultures) (day 2 – step 5) each add 100mM IPTG, these will become the IPTGex30, IPTGex60, IPTGex120 cultures
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*To 3 of 4 cultures (day 2 – step 6) each add excessive amount of Arabinose (need to ask Glenn how much he used), these will become the ARAex30, ARAex60, ARAex120 cultures
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*To 3 of 4 cultures (day 2 – step 6) each add excessive amount of Arabinose, these will become the ARAex30, ARAex60, ARAex120 cultures
*From all 8 cultures (IPTG-, IPTGex30, IPTGex60, IPTGex120, ARA-, ARAex30, ARAex60, ARAex120) take 1.5 ml sample and put on ice (-20 C) (IPTG t0 & ARA t0)
*From all 8 cultures (IPTG-, IPTGex30, IPTGex60, IPTGex120, ARA-, ARAex30, ARAex60, ARAex120) take 1.5 ml sample and put on ice (-20 C) (IPTG t0 & ARA t0)
*At 30 min take for all 8 cultures (IPTG-, IPTGex30, IPTGex60, IPTGex120, ARA-, ARAex30, ARAex60, ARAex120) a 1.5 ml sample and put on ice (-20 C)  (IPTG t30 & ARA t30)
*At 30 min take for all 8 cultures (IPTG-, IPTGex30, IPTGex60, IPTGex120, ARA-, ARAex30, ARAex60, ARAex120) a 1.5 ml sample and put on ice (-20 C)  (IPTG t30 & ARA t30)

Latest revision as of 03:55, 27 September 2012