Team:Exeter/lab book/3gip/wk7

Operon Construction  |  Glycobase

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Tuesday 21st August

Morning

•Gel Electrophoresis

The 3A assembly from last week 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

•Two Step Phusion PCR

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 overnight so a gel could be run in the 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

•Transformation

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 concentrations 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:

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 as did the wbbC gene from genomic DNA. The genomic DNA may not hold the wbbC gene.

Afternoon

Made competent cells.

40ml of LB was inoculates in a 250ml Monod flask with 0.4ml of the overnight culture. Growth at 37°C and 200rpm until A650=0.4-0.5.

The culture was transferred to a 40ml Falcon sterile tube and harvested by centrifugation at 8,000rpm for 8 minutes at 4°C.

The pellet was drained and resuspended in 4ml of TF-2 solution.

This was then placed on ice for 15 minutes and then spun again as above.

The pellets were drained and resuspended in 4ml of TF-2.

The competent cells were then spit into 0.2ml aliquots and frozen at -70°C immediately.