Team:Exeter/lab book/1gp/wk4

Operon Construction  |  Glycobase

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• Genomic extraction from E.coli BL21(DE3)

• PCR amplification of WbbC from E.coli BL21(DE3) using two-step PCR

• Gel Electrophoresis to check fragment size of WbbC PCR product

The following concentration (in ng/µL) obtained for the BL21(DE3) genomic DNA were:

o 1. - 7.8.

o 2. - 6.6.

Gel Electrophoresis did not show any correct PCR products (expected: 1113bp band) and it was discovered that the reverse primer was incorrect and had actually amplified a shorter section of WbbC, rather than the whole gene. Thus the correct reverse primer was ordered. Lane 1 = DNA hyperladder, Lane 2 = WbbC attempt from genomic DNA template 1, Lane 3 = WbbC attempt from genomic DNA template 2.

Tuesday 31st July (15.00)

• IDT re-suspension and transformation of WbbC(with point mutation)

Wednesday 1st August (15.00)

• Adding cultures with WbbC(with point mutation) plasmids into liquid medium and incubation overnight

Protocol was followed using ampicillin for WbbC(with point mutation) plasmids as the selection antibiotic.

Thursday 2nd August (9.00)

• Mini-Prepping of WbbC(with point mutation) plasmid

• Gel Electrophoresis to check fragment sizes of WbbC(with point mutation) plasmid

The following concentrations (in ng/µL) was obtained:

o WbbC(with point mutation) - 181.6.

Gel Electrophoresis showed that WbbC(with point mutation) did not work because whilst there was a band, it was the wrong size (expected: 1113bp). This procedure was repeated again. Lane 1 = DNA hyperladder, Lane 2 = Mini-prepped WbbC.