Team:Nevada/Protocols

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(Gibson Assembly)
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==Gibson Assembly==
==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. Gibson Assembly Reaction Setup: Amount per Reaction
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:::#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. Gibson Assembly Reaction Setup: Amount per Reaction
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PCR Fragment(s)+linearized vector 2-10 μl
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PCR Fragment(s)+linearized vector2-10 μl
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Gibson Assembly Master Mix (2X) 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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Deionized H2O XX μl
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Total Volume 20 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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:::#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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:::#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.
:::#Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases (for further details see FAQ section).
:::#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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:::#Transform NEB 5-alpha Competent E. coli cells (provided with the kit) with 2 μl of the assembly reaction, following the transformation protocol.
==Transformation==
==Transformation==

Latest revision as of 04:22, 28 September 2013