Team:Exeter/lab book/gibs/wk9
From 2012.igem.org
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Operon Construction: 3rd - 7th September 2012 **Monday 3.9.12**Gibson PCR again Fragments and concentrations as 30.8.12 PCR setup: 98 °C for 30s 98 °C for 10s, 69 °C for 30s, 72 °C for 20s – X 5 98 °C for 10s, 72 °C for 30s – X 25 72 °C for 10minutes Hold temp. 4 °C Ran gel – only wfcA 1 & 2 worked **Tuesday 4.9.12** Mega-miniprep day: Due to various iGEM commitments, an overwhelming number of minipreps was required. LB broth was inoculated 3.9.12 - Four colonies from each plate from all transformations from 21.08.12 onwards were put in broth and miniprepped (see Freddie’s lab book for 3 gene inducible plasmid for details)
Miniprep according to **Wednesday 5.9.12** Used nanodrop spectrophotometer to find concentrations from minipreps the previous day Gibson PCR of Pbad, wbnJ, wbbC and wbnK 2. DNA 1 µl, water 33 µl 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 10 minutes Hold temp. 4 °C Gel run – no success **Thursday 6.9.12** Gibson PCR using Immomix red (2x). Pbad, wbnJ, wbbC, wbnK 2. Immomix red 25 µl DNA 1 µl Primer 1 – 2 µl Primer 2 – 2 µl Immomix red 2x – 25 µl Water - 20 µl PCR setup: 95 °C for 10 minutes 95 °C for 10s, 55 °C for 30s, 72 °C for 30s – X 25 Hold temp. 4 °C **Friday 7.9.12** Ran gel from previous PCR – wbbC worked (although significant blurring – fewer PCR steps may have helped.) Gibson PCR of Pbad, wbnJ and wbnK 2 using immomix red again Volumes as on 6.9.12 PCR setup: 95 °C for 10 minutes 95 °C for 10s, 65 °C for 30s, 72 °C for 30s – X 5 4 °C hold step Ran gel – no success |
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