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Team:Exeter/lab book/3gip/wk7
From 2012.igem.org
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The 3-Gene Inducible Plasmid: 20th - 24th August 2012 Tuesday 21st August Morning •Gel Electrophoresis Last weeks 3A assembly was run to check fragment sizes after digestion using the EcoR1 and Pst1 enzymes wfcA + BBa_B0014 wbnJ + BBa_B0014 BBa_B0034 wbbC + BBa_B0014 Afternoon The reaction was set up using the Biolabs Phusion PCR protocol. Two reactions were set up: Reaction 1: fadR control Reaction 2: genomic DNA hopefully containing the wbbC gene The PCR mix was stored at -20°C over night so a gel could be run the next morning. Wednesday 22nd August Morning A 1% agarose gel was prepared. 1μl of loading buffer was added to 5μl of the PCR mix and this was loaded onto the gel and run for 20 minutes. The fadR control showed a strong band but the wbbC gene showed nothing. •Two Step Phusion PCR using an initial lower annealing temperature The reaction was set up using the Biolabs Phusion PCR protocol. Two reactions were set up: Reaction 1: wbbC (sequenced with mutation) control Reaction 2: genomic DNA hopefully containing the wbbC gene A 1% agarose gel was prepared. 1uμl of loading buffer was added to 5μl of the PCR mix and this was loaded onto the gel and run for 20 minutes. The wbbC control showed a strong band but the wbbC genomic gene showed nothing. We know that the primers work with wbbC. Afternoon hasA + BBa_B0014 in pSB1T3 amyA (cyclodextran gene) + BBa_B0014 in pSB1T3 sacB + BBa_B0014 in pSB1T3 Using Invitrogen TOP10 competent cells split into 2 eppendorfs containing 25μl each. Spread on two tetracycline plates for each transformation using 20μl and 150μls respectively. Transformed for Becca. See Showcasing Polysaccharide Production: 20th - 24th August 2012, Thursday 23/08/12 Thursday 23rd August Morning • Two Step Phusion PCR using serial dilution of genomic DNA The reaction was set up using the Biolabs Phusion PCR Protocol. Two reactions were set up: Reaction 1: wbbC (sequenced with mutation) control Reaction 2: genomic DNA hopefully containing the WbbC gene The genomic DNA was nanodropped and from this four different concentraitons prepared. Four reaction tubes containing 1μl, 0.8μl, 0.5μl and 0.1μl were used for this PCR reaction.A 1% agarose gel was prepared. 1μl of loading buffer was added to 5μl of the PCR mix and this was loaded onto the gel and run for 20 minutes. The wbbC control showed a strong band but the wbbC gene showed nothing. We know that the primers work with wbbC. Afternoon •Competent Cells Before the transformation started, 1μl of cells from two tubes were pipetted onto agar plates containing no antibiotic and incubated overnight at 37°C. Friday 24th August Morning •Three Step Phusion PCR using a 10 fold dilution The reaction was set up using the Biolabs Phusion PCR Three Step protocol. 28 reactions were set up reactions were set up: wbbC (sequenced with mutation) control as a positive control at either end of the PCR gradient Mastermix as a negative control at either side of the PCR gradient genomic DNA hopefully containing the wbbC gene across the PCR gradient using the 10 fold dilution. A 1% agarose gel was prepared. 1ul of loading buffer was added to 5ul of the PCR mix and this was loaded onto the gel and run for 20 minutes. The wbbC control showed a strong band, the negative control showed nothing and the wbbC gene from genomic DNA showed nothing. The genomic DNA may not hold the wbbC gene. Afternoon Made competent cells in the afternoon following the protocol exactly. |
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