Team:Exeter/lab book/3gip/wk5

Operon Construction  |  Glycobase

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Monday 6th August

Morning

•3A Assembly

BBa_J23119 + BBa_B0034 into pSB1C3

BBa_B0034 + wbbC into pSB1C3

Performed with the NEB protocol but using the Linearized Plasmid Protocol from the parts registry. The volume of digestion was changed from 50ul to 20ul 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.

A mistake was made here. The remaining mastermix from the 3A assembly from the previous week was used and therefore the enzymes were not fresh.

After ligation the samples were stored at -20°C.

Afternoon

• IDT re-suspension and Transformation of wbnK was started in the afternoon and then handed over to Alex and Becca.

Tuesday 7th August

Afternoon

Parts Registry: Requesting Parts

An Agar stab of BioBrick BBa_K322921 was received in the morning. In the afternoon the BioBrick was streaked out on an ampicillin plate using an inoculating loop in the sterile hood.

Wednesday 8th August

Afternoon

•Transformation

BBa_J23119 + BBa_B0034 in pSB1C3

BBa_B0034 + wbbC in pSB1C3

Using Invitrogen TOP10 competent cells split into 2 eppendorfs containing 25μl each.

Spread on two chloramphenicol plates for each transformation using 20μl and 100μls respectively.

Thursday 9th August

Morning

•3A Assembly

BBa_B0034 + wfcA into pSB1C3

Dilutions and timing were worked out but went to help with another 3A assembly already in progress. See Showcasing Polysaccharide Production Thursday 09/08/12

Afternoon

•Transferring colonies to liquid medium

Cells transformed with BBa_J23119 + BBa_B0034 and BBa_B0034 + wbbC were added into liquid medium and incubated overnight.

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

Friday 10th August

Morning

•Miniprepping

BBa_J23119 + BBa_B0034

BBa_B0034 + wbbC

Using the changed protocol. The tubes were centrifuged for 10 minutes to start to ensure a good pellet formed.

After the neutralisation solution had been added, the eppendorfs were centrifuged for 10 minutes rather than 5 in order to get a better pellet formation.

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

We had some pipetting trouble when adding the neutralisation solution. The delay caused by this may have affected our results.

BBa_J23119 + BBa_B0034 was nanodropped and recorded very poor concentrations.

BBa_B0034 + wbbC was nanodropped and recorded very poor concentrations.

• Gel Electrophoresis

Run to check fragment sizes of BBa_B0034 + wbbC after digestion using the EcoR1 and Pst1 enzymes

Gel Electrophoresis showed a band at the correct size.

•3A Assembly

BBa_J13002 + wbnK_BBa_B0014 in pSB1T3

wfcA + BBa_B0014 in pSB1T3

wbnJ + BBa_B0014 in pSB1T3

BBa_B0034_wbbC + BBa_B0014 in pSB1T3

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.

The ligation was stored at -20°C for the weekend and transformed on Monday.