Team:Nevada/Protocols

From 2013.igem.org

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==Qiagen Miniprep kit==
==Qiagen Miniprep kit==
:::www.qiagen.com/hb/qiaprepminiprep<br/>
:::www.qiagen.com/hb/qiaprepminiprep<br/>
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==Gibson Assembly==
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:::#1.Please note that this protocol has been optimized for the assembly and cloning of one or two fragments into any vector. For the cloning of larger assemblies, we recommend using the standard protocol provided with the Gibson Assembly Master Mix
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Gibson Assembly Reaction Setup
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Amount per Reaction
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PCR Fragment(s)+linearized vector 2-10 μl
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Gibson Assembly Master Mix (2X) 10 μl
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Deionized H2O XX μl
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Total Volume 20 μl
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:::#2.Optimized cloning efficiency requires about 25–100 ng of vector and at least 2-fold excess inserts. Use 5–10X more insert if the size is less than 200 bp. Total volume of unpurified PCR fragments in Gibson Assembly reaction should not exceed 20%.
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:::#3.Incubate samples in a thermocycler at 50°C for 15 minutes. After incubation, store the samples on ice or at -20°C for subsequent transformation.
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Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases (for further details see FAQ section).
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:::#4.Transform NEB 5-alpha Competent E. coli cells (provided with the kit) with 2 μl of the assembly reaction, following the transformation protocol.
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==Transformation==
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:::#1.Thaw competent cells on ice.
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:::#2.Add 2 μl of the chilled assembly product to the competent cells. Mix gently by pipetting up and down or by flicking the tube 4–5 times. Do not vortex.
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:::#3.Place the mixture on ice for 30 minutes. Do not mix.
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:::#4.Heat shock at 42°C for 30 seconds. Do not mix.
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:::#5.Transfer tubes to ice for 2 minutes.
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:::#6.Add 950 μl of room-temperature SOC media to the tube.
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:::#7.Incubate the tube at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
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:::#8.Warm selection plates to 37°C.
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:::#9.Spread 100 μl of the cells onto the selection plates. Use Amp plates for positive control sample.
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:::#10.Incubate overnight at 37°C.

Revision as of 04:19, 28 September 2013