Team:Exeter/lab book/3gip/wk4

Operon Construction  |  Glycobase

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Monday 30th July

Afternoon

• Adding cultures

Cells transformed with BBa_I0500 were added into liquid medium and incubated overnight.

Protocol was followed using kanamycin for the BioBrick part as the selection antibiotic.

Tuesday 31th July

Morning

• Mini-Prepping

BBa_I0500

BBa_I0500 was nanodropped and recorded poor concentrations.

•Gel Electrophoresis

Run to check fragment sizes of BBa_I0500 after digestion using the EcoR1 and Pst1 enzymes

Gel Electrophoresis showed a faint band at the correct size.

• Sequencing

BBa_K094120_ BBa_B0034 was sent off for sequencing using the primers VF2 and VR.

•3A assembly:

BBa_I0500 + BBa_B0034

Performed with the NEB protocol but using the Linearized Plasmid Protocol from the parts registry. The volume of digestion was changed from 50μl to 20μl by adding less water so as to increase the DNA concentration. As described in the Linearized Plasmid Protocol, a mastermix was made and 4μl of this was added to 4μl of destination plasmid for the digestion.

Afternoon

•Transformation:

BBa_I0500 + BBa_B0034

Using Invitrogen TOP10 competent cells.

Spread on two kanamycin plates with 20μl and 100μls respectively.

Wednesday 1st August

Morning

Only a few small colonies were found on the plate for BBa_I0500 + BBa_B0034 this morning.

Afternoon

•Transformation:

Constitutive promoter BBa_J23119, 2012 Distribution Kit Plate 1, 18A

Using Invitrogen TOP10 competent cells.

Spread on two ampicillin plates with 20μl and 100μls respectively.

•Adding cultures

Cells transformed with BBa_I0500 + BBa_B0034 were added into liquid medium and incubated overnight

Protocol was followed using kanamycin for the BioBrick part as the selection antibiotic.

Thursday 2nd August

Morning

Good colonies were found on the 150μl BBa_J23119 plates this morning. They were placed in the fridge ready for later.

•Mini-Prepping of BBa_I0500 + BBa_B0034 by Chris and Sophie our work experience students

BBa_I0500 + BBa_B0034 was nanodropped and showed no DNA. A gel was run to test this and it confirmed no DNA was present.

•Gel Electrophoresis was run to check whether any DNA was present and to confirm the nanodrop readings after digestion using the EcoR1 and Pst1 enzymes. The gel was set up and run as 100ng of DNA. A 1% agarose gel was used.

Gel Electrophoresis showed no bands.

Afternoon

Streak plates were then made with the remaining broth from the BBa_I0500 + BBa_B0034 culture growth and incubated overnight at 37°C.

•Adding cultures

Cells transformed with BBa_J23119 were added to liquid medium and incubated overnight.

Protocol was followed using ampicillin for the BioBrick part as the selection antibiotic.

Friday 3rd August

Morning

•Mini-Prepping of BBa_J23119

The protocol was changed slightly. After the neutralisation solution had been added, the eppendorfs were centrifuged for 10 minutes rather than 5 in order to obtain a better pellet formation.

40ul of water was added instead of 50µl in order to make sure the concentration of DNA increased.

BBa_I0500 was nanodropped and recorded good concentrations.

The promoter was too small to show up on a 1% gel electrophoresis.