Team:Cambridge/LabBook/Week4

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Inoculated single bacterial (TOP10 with BBa-J13002 and TOP10 with BBa_I712019 individually) colony in 5ml LB, incubated at 37°C with shaking (180rpm) overnight
Inoculated single bacterial (TOP10 with BBa-J13002 and TOP10 with BBa_I712019 individually) colony in 5ml LB, incubated at 37°C with shaking (180rpm) overnight
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==Wednesday==
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===13. Experiment: Measuring competency (W3110 hns 93-1, BW25113 Δhns::kan, W3110 hns-205::Tn10 Tet<sup>R</sup>, GM230 hns-205::Tn10 Tet<sup>R</sup>) (Theo & Hannah)===
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Followed protocol under 'TOP10 chemically competent cells' from OpenWetware
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Competent cells from all 4 different strains taken out of -80°C freezer and thawed on ice. 1ml pipette tips  cut with scissors dipped in ethanol and flamed. For each strain 50μl of cells were transferred to a separate 1.5ml Eppendorf tube. 1μl pUC19 (standard plasmid) was added to each 50μl of cells. Held on ice for 30 min. Heat shocked at 42°C for 60s (water bath). On ice for ~2min. 250μl SOC was added to each of 4 tubes. Each Eppendorf tube was put in a 12ml falcon with tape over the top and incubated at 37°C for 1h with rotation. 20μl of each of the 4 different transformed strains were plated on LB agar plages with Amp. Colonies were grown overnight at 37°C.
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===14. Experiment: Making competent and transforming <i>E. coli</i>/pHK555 (Peter & Paul)===
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50μl EZ Comp  buffer transferred to Eppendorf tube and placed on ice. Single <i>E. coli</i>/pHK555 colony resuspended in EZ Comp buffer(2x single colony in 50μl EZ each, 1 streak of colonies in another 50μl EZ). Vortex if needed to suspend clumps. 3μl of pHK724 plasmid added to solution, mixed and incubated on ice for 20 min.
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Heaat shocked at 42°C for 90s (water bath). Incubated at RT for 5 min. 1ml LB added. Eppendorf tubes put in 12ml falcon tubes with tape over top. Incubated at 37°C for 1h with rotation. Plated 100μl on LB agar plates with Cm and Amp. Incubated at 30°C for 48h.
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===15. Experiment BioBrick Standard Assembly (Emily & Bill, Paul & Anja)===
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Took overnight cultures of
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# TOP10 with BBa_J13002 (plasmid with promoter + rbs)
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# TOP10 with BBa_I712019 (plasmid with firefly luciferase)
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====Plasmid DNA Purification==== following the "QIAprrep Spin Miniprep Kit using a Microcentrifuge" protocol.
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====Restriction enzyme digest====
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Prepared at RT in the listed order:
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{| class="wikitable"
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|-
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!
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! promoter + rbs
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! luciferase
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|-
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| nuclease-free H<sub>2</sub>O
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| 15μl
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| 14μl
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|-
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| 10x Fast Digest Buffer
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| 2μl
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| 2μl
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|-
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| Plasmid DNA
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| 2μl
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| μl
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|-
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| FD enxyme SpeI
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| 1μl
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| 1μl
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|-
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| FD enzyme XbaI
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| -
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| 1μl
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|}
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It was checked (Nanodrop!) that 2μl of plasmid DNA would not contain more than 1μg of DNA. The reaction mixtures were mixed gently (flick tube) and spun in the microcentrifuge for 15s (13,000 rpm). It was then incubated at 37°C (water bath) for 30 min.
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====Gel Electrophoresis====
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An E-gel EX Agarose 1% was mounted on the transilluminator. Nanodrop readings were taken for both restriction enzyme digests:
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*Plasmid with promoter + rbs: 21.4 ng/μl => 5μl gives 100ng
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*Plasmid with luciferase: 19.7ng/μl => 5μl gives 100ng
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For each digest the following mixture was made up
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* 3μl 6X orange loading Dye
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* 5μl plasmid DNA (restriction digest)
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* 12μl deionised H<sub>2</sub>O
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Gel was loaded according to the scheme  below:
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{| class="wikitable"
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|-
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| Easyladder II
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| promoter + rbs
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| promoter + rbs
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| luciferase
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| luciferase
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|-
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| 10μl
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| 20μl
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| 20μl
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| 20μl
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| 20μl
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|}
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Revision as of 13:23, 9 August 2010

Retrieved from "http://2010.igem.org/Team:Cambridge/LabBook/Week4"