Team:USP-Brazil/GaTE

From 2013.igem.org

(Difference between revisions)
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PCR conditions tested for Q5 High Fidelity 2X Master Mix:
PCR conditions tested for Q5 High Fidelity 2X Master Mix:
<ul>
<ul>
-
<li>annealing temperature of 53°C for pAOX1 (7/06/13 gradient PCR)) along with the use of 1/10 yeast gDNA dilution
+
<li>annealing temperature of 53°C for pAOX1 (7/06/13 gradient PCR) along with the use of 1/10 yeast gDNA dilution
<li>67°C annealing  temperature and 0.5 uL of 1/100 dilution RFP template
<li>67°C annealing  temperature and 0.5 uL of 1/100 dilution RFP template
</ul></p>
</ul></p>
 +
<p>Found out best conditions to amplify pAOX1 (which are shown in the end of the lab diary) <span style="color:red;"><b>(TA FALTANDO A TEMPERATURA!)</b></span> <br/>
 +
pPIC9K digest with EcoRI and NotI and kept frozen<br/>
 +
Gibson assembly using NEB Gibson Assembly cloning kit of pAOX1, RFP and EcoRI/NotI digested pPIC9K<br/>
 +
Transformation of DH10B E. coli with pPIC9K using electroporation<br/>
 +
Select, inoculate, and incubate colonies from Gibson plates (pPIC9K-pAOX1-RFP) and pPIC9K plates<br/>
 +
Plasmid extraction of transformed colonies<br/></p>
 +
<p style="color:red;"><b>(falar o que deu errado)</b></p>
 +
 +
<p><b>17 Jun – 21 Jun</b><br/>
 +
Transformation of DH10B and NEB 5-alpha competent E. coli with Gibson assembly product using electroporation and heat shock, respectively<br/>
 +
Plate transformed cells<br/>
 +
Select, inoculate, and incubate colonies from heat shock and electroporation Gibson assembly plates<br/>
 +
Plasmid extraction and plasmid digestion gel (Gibson assembly with NotI enzyme)<br/>
 +
RFP PCR amplified at 69 °C annealing temperature</p>
 +
<p style="color:red;"><b>(falar o que deu errado na extração ou no que tiver dado errado)</b></p>
 +
 +
<p><b>28 Jun – 30 Jun</b><br/>
 +
RFP PCR using new Gibson assembly compatible primers <br/>
 +
DNA gel to check<br/>
 +
Gibson assembly of pAOX1, RFP, and EcoRI/NotI digested pPIC9K and transformation using NEB 5-alpha competent cells (heat shock) and DH10B (electroporation), plating 100 uL and 900 uL for each on ampicillin plates<br/>
 +
Select, inoculate, and incubate from plates <br/>
 +
Spin down and freeze samples</p>
 +
 +
<p><b>1 Jul – 5 Jul</b><br/>
 +
Plasmid extraction from inoculated colonies<br/>
 +
Plasmid digestion using NotI<br/>
 +
DNA agarose gel to check. <span style="color:red;"><b>(FALAR O QUE DEU NO GEL)</b></span><br/>
 +
Plasmid digestion of heatshock transformed samples<br/>
 +
PCR of pAOX1 and RFP followed by purification using NucleoSpin PCR Clean-up kit<br/>
 +
Gel of PCR products and digests to check<br/>
 +
Quantification of RFP PCR product and pPIC9K previously digested by using gel and Nanodrop</p>
 +
 +
<p style="color:red;"><b>Table 1</b></p>
 +
 +
<p>NucleoSpin Clean-up of pPIC9K overnight digest<br/>
 +
DNA gel of pAOX1 PCR and pPIC9K digest purification<br/>
 +
Repeat pAOX1 PCR to obtain more pAOX1 sample<br/>
 +
Combine pAOX1 PCR products and purify with NucleoSpin Clean-up<br/>
 +
DNA gel of purified pAOX1 and pPIC9K digested/undigested<br/>
 +
pAOX1 PCR using 2 uL of 1/10 yeast genomic DNA with 50°C and 57°C annealing temperatures<br/>
 +
Overnight digest of pPIC9K<br/>
 +
pAOX1 PCR at 60°C annealing temperature<br/>
 +
DNA gel of 60°C annealing PCR product; 5 uL PCR product and 2 uL loading buffer<br/>
 +
Pool both paOX1 PCR products together and run an agarose gel <br/>
 +
Run pPIC9K overnight digestion on gel<br/>
 +
Gel extraction of pAOX1 and pPIC9K</p>
 +
 +
 +

Revision as of 07:33, 27 September 2013

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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

Found out best conditions to amplify pAOX1 (which are shown in the end of the lab diary) (TA FALTANDO A TEMPERATURA!)
pPIC9K digest with EcoRI and NotI and kept frozen
Gibson assembly using NEB Gibson Assembly cloning kit of pAOX1, RFP and EcoRI/NotI digested pPIC9K
Transformation of DH10B E. coli with pPIC9K using electroporation
Select, inoculate, and incubate colonies from Gibson plates (pPIC9K-pAOX1-RFP) and pPIC9K plates
Plasmid extraction of transformed colonies

(falar o que deu errado)

17 Jun – 21 Jun
Transformation of DH10B and NEB 5-alpha competent E. coli with Gibson assembly product using electroporation and heat shock, respectively
Plate transformed cells
Select, inoculate, and incubate colonies from heat shock and electroporation Gibson assembly plates
Plasmid extraction and plasmid digestion gel (Gibson assembly with NotI enzyme)
RFP PCR amplified at 69 °C annealing temperature

(falar o que deu errado na extração ou no que tiver dado errado)

28 Jun – 30 Jun
RFP PCR using new Gibson assembly compatible primers
DNA gel to check
Gibson assembly of pAOX1, RFP, and EcoRI/NotI digested pPIC9K and transformation using NEB 5-alpha competent cells (heat shock) and DH10B (electroporation), plating 100 uL and 900 uL for each on ampicillin plates
Select, inoculate, and incubate from plates
Spin down and freeze samples

1 Jul – 5 Jul
Plasmid extraction from inoculated colonies
Plasmid digestion using NotI
DNA agarose gel to check. (FALAR O QUE DEU NO GEL)
Plasmid digestion of heatshock transformed samples
PCR of pAOX1 and RFP followed by purification using NucleoSpin PCR Clean-up kit
Gel of PCR products and digests to check
Quantification of RFP PCR product and pPIC9K previously digested by using gel and Nanodrop

Table 1

NucleoSpin Clean-up of pPIC9K overnight digest
DNA gel of pAOX1 PCR and pPIC9K digest purification
Repeat pAOX1 PCR to obtain more pAOX1 sample
Combine pAOX1 PCR products and purify with NucleoSpin Clean-up
DNA gel of purified pAOX1 and pPIC9K digested/undigested
pAOX1 PCR using 2 uL of 1/10 yeast genomic DNA with 50°C and 57°C annealing temperatures
Overnight digest of pPIC9K
pAOX1 PCR at 60°C annealing temperature
DNA gel of 60°C annealing PCR product; 5 uL PCR product and 2 uL loading buffer
Pool both paOX1 PCR products together and run an agarose gel
Run pPIC9K overnight digestion on gel
Gel extraction of pAOX1 and pPIC9K

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