Team:Exeter/lab book/3gip/wk5

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ExiGEM2012 Lab Book 3GP wk5


Protocols \| Single Gene Plasmids and Enzyme Characterisation \| Showcasing Polysaccharide Production \| The 3-Gene Inducible Plasmid Operon Construction \| Glycobase
9th - 13th July - 16th - 20th July - 23rd - 27th July - 30th July - 3rd August - 6th - 10th August - 13th - 17th August - 20th - 24th August - 27th - 31st August - 3rd - 7th September - 10th - 14th September - 17th - 21st September
	The 3-Gene Inducible Plasmid: 6th - 10th August 2012 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. Remaining mastermix from last weeks 3A assembly was used and therefore the enzymes were not fresh. After ligation the samples were stored at -20°C. Afternoon • IDT re-suspension antransformation of wbnK was started in the afternoon and then handed over to Alex and Becca. Tuesday 7th August Afternoon http://partsregistry.org/Help: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. https://2012.igem.org/Team:Exeter/lab_book/novpol/wk5 Afternoon • Adding cultures 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 •Mini-Prepping 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 40μl in order to make sure the concentration of DNA increased. We had some pipetting trouble when adding the neutralisation solution. The delay cause by this may have affected 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.