Team:USP-Brazil/GaTE

From 2013.igem.org

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<h2>GaTE Lab</h2>
<h2>GaTE Lab</h2>
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<p>Text here.</p>
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<p><b>22 May – 24 May</b><br/>
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RFP and pAOX1 were obtained from <span style="color:red;"><b>?????</b></span> <br/>
 +
Plasmid BBa_E1010 that contains RFP gene was transformed into DH10B and plated on kanamicin plates from which a single colony was inoculated for miniprep.<br/>
 +
Plasmid extraction successful was detected by running an agarose gel and its quantification was determined by nanodrop.<br/>
 +
RFP gene was amplified by Gradient PCR for further assembly. It was performed using 1/10 and 1/100 dilutions of a RFP sample 1540 ng/uL.<br/>
 +
1/100 dilution at 65°C appears like optimal PCR conditions for RFP PCR. </p>
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<p><b>3 Jun – 7 Jun</b><br/>
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To verify in which annealing temperature the pAOX1 promoter right size fragment is obtained a Gradient PCR was performed. pAOX1 amplification was executed by using GoTaq Polimerase <br/>
 +
PCR was performed with pAOX1 primers dilution from 100 mM (correspondent to 1/10 dilution from stock) to 10 mM (correspondent to 1/100 dilution from stock), 3 different yeast gDNA concentration (750 ng/uL,1606 ng/uL and 1303 ng/uL) and in a 53°C to 65°C temperature range.<br/>
 +
Best conditions were detected by analysing Gradient PCR on a gel run: 1/100 dilution at 53°C  appears most reliable, but 1/10 dilution is most pronounced </p>
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<p><b>10 Jun – 14 Jun</b><br/>
 +
pAOX1 was amplified with less quantity of Q5 High Fidelity 2X Master Mix and longer thermocycling extension to compare to 24/5/13 run using 1/100 dilution of RFP template<br/>
 +
PCR conditions tested for Q5 High Fidelity 2X Master Mix:
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<ul>
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<li>annealing temperature of 53°C for pAOX1 (7/06/13 gradient PCR)) along with the use of 1/10 yeast gDNA dilution
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<li>67°C annealing  temperature and 0.5 uL of 1/100 dilution RFP template
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</ul></p>
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</div>
</div>

Revision as of 07:16, 27 September 2013

Template:Https://2013.igem.org/Team:USP-Brazil/templateUP

Notebook

GaTE Lab

22 May – 24 May
RFP and pAOX1 were obtained from ?????
Plasmid BBa_E1010 that contains RFP gene was transformed into DH10B and plated on kanamicin plates from which a single colony was inoculated for miniprep.
Plasmid extraction successful was detected by running an agarose gel and its quantification was determined by nanodrop.
RFP gene was amplified by Gradient PCR for further assembly. It was performed using 1/10 and 1/100 dilutions of a RFP sample 1540 ng/uL.
1/100 dilution at 65°C appears like optimal PCR conditions for RFP PCR.

3 Jun – 7 Jun
To verify in which annealing temperature the pAOX1 promoter right size fragment is obtained a Gradient PCR was performed. pAOX1 amplification was executed by using GoTaq Polimerase
PCR was performed with pAOX1 primers dilution from 100 mM (correspondent to 1/10 dilution from stock) to 10 mM (correspondent to 1/100 dilution from stock), 3 different yeast gDNA concentration (750 ng/uL,1606 ng/uL and 1303 ng/uL) and in a 53°C to 65°C temperature range.
Best conditions were detected by analysing Gradient PCR on a gel run: 1/100 dilution at 53°C  appears most reliable, but 1/10 dilution is most pronounced

10 Jun – 14 Jun
pAOX1 was amplified with less quantity of Q5 High Fidelity 2X Master Mix and longer thermocycling extension to compare to 24/5/13 run using 1/100 dilution of RFP template
PCR conditions tested for Q5 High Fidelity 2X Master Mix:

  • annealing temperature of 53°C for pAOX1 (7/06/13 gradient PCR)) along with the use of 1/10 yeast gDNA dilution
  • 67°C annealing  temperature and 0.5 uL of 1/100 dilution RFP template

Template:Https://2013.igem.org/Team:USP-Brazil/templateDOWN