Team:Exeter/lab book/gibs/wk8

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ExiGEM2012 Lab Book Gibs wk6


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
	Operon Construction: 27th - 31st August 2012 Tuesday 28.8.12 EcoRI and PstI digest of genes (wbnJ, wbnK, wbbC(d) & wfcA)- for checking on gel Digestion protocol Water to 20 µl 3A assembled genes to upstream RBS (BBa_B0034) BBa_B0034 + wbnJ in pSB1K3 BBa_B0034 + wbnK in pSB1K3 BBa_B0034 + wfcA in pSB1K3 BBa_B0034 + wbbC(1) in pSB1K3 BBa_B0034 + wbbC(2) in pSB1K3 BBa_B0034 + wbbC(3) in pSB1K3 3A assembly Digest concentrations and volumes were altered like so: DNA – 500 ng Enzyme A – 0.5 µl Enzyme B – 0.5 µl NEBuffer 2 – 2 µl BSA – 0.2 µl Water – to 20 µl Ran gel of PCR product from 24.8.12 (not successful PCR) PCR using BL21 genomic DNA (extracted previously by Alex B. and Liam) Control - 0.3 µl DNA, 34.7 µl water Genomic – 5 µl DNA, 31 µl water 3 step PCR 2 step PCR PCR setup 98°C for 30s 98°C for 10s, 50°C for 30s, 72°C for 20s – X 5 98°C for 10s, 72°C for 30s – X 25 72°C for 10mins Hold temp. 4 °C Ran PCR gel – success! BL21 genomic DNA was the key. Transformed extra vectors from plates – standard procedure BioBrick extraction Transformation of competent cells Wednesday 29.8.12 PCR purification of wbbC PCR product *Step 1* 225 µl buffer PB added to 45 µl product *Additional step after 5* 750 µl PE added. Centrifuged 1 min, 13000 rpm, rt. Discarded flow through *Step 7* 30 µl water added. Stood for 1 minute. 20 µl extra water added (water was not on centre of membrane) Stored at -20 °C PCR for Gibson assembly Fragments were primed with overlap primers for making three operon constructs Fragments and volumes (to get 50 ng/µl): Pbad (large) - 1 µl wbnJ – 0.15 µl wbbC - 1 µl wfcA for operons 1 and 3 – 0.15 µl wbnK for 2 and 3 – 0.19 µl Double terminator for 1, 2 and 3 (2 and 3 will be the same, it joins to wbnK in both) – 1 µl (N.B. dilutions of DNA were made to reach appropriate concentration – these dilutions were used for all following PCR reactions which is why each following PCR write-up states 1 µl of DNA was used) water to 50 µl 3 step PCR 2 step PCR PCR setup: 98°C for 30s 98 °C for 10s, 50°C for 30s, 72°C for 20s – X 5 98°C for 10s, 72°C for 30s – X 25 72°C for 10mins Hold temp. 4 °C Thursday 30.8.12 Miniprep of vectors according to Fermentas protocol *Step 745 µl water added, followed by another 5 µl Gibson PCR Pbad L, wbbC, wfcA* 1 & 2, wbnK 1 DNA 1 µl, water 33 µl 3 step PCR PCR setup: 98°C for 30s 98 °C for 10s, 50°C for 30s, 72°C for 20s – X 5 98 °C for 10s, 70°C for 30s, 72°C for 20s – X 5 98 °C for 10s, 72°C for 30s – X 20 72 °C for 10mins Hold temp. 4 °C Ran gel – no luck