[[GoldenGateAssemblyProtocolsMainPage][Back to Golden Gate Protocols]] ---+ Second Stage Assembly Second Stage assemblies are used to build plasmids out of multiple transcriptional units ([[http://barricklab.org/twiki/bin/view/Lab/ProtocolsBTKAssembleSingleTUPlasmid Protocol found here]]). Occasionally this assembly can be used as a second step to building troublesome first stage plasmids - the first stage plasmid is assembled in a high copy/easier to work with backbone then moved to lower copy/more difficult to work with backbone. This assembly makes use of the BsmBI enzyme as described below. Protocol source: NEB ([[https://www.neb.com/protocols/2020/01/15/golden-gate-assembly-protocol-for-using-neb-golden-gate-assembly-kit-bsmbi-v2-neb-e1602]]) ---+++ Assembly reaction [[Old_Golden_Gate_Assembly][Access the old non-kit golden gate assembly protocols here]] Total volume will be 20 μL; you will need 50 fmol of each transcriptional unit plasmid for this assembly. Various backbones are regularly used in this step - if toxicity issues may be a factor, lower copy number vectors are recommended. * 50 fmol of each transcriptional unit/backbone = 650 * insert length * 50x10^-6 = *X ng* * 2 μL of NEB 10× T4 DNA ligase buffer * 1 μL of NEB Golden Gate Enzyme Mix BsmBI-v2 * x μL water up to 20 μL total. For easy part volume calculation: [[%ATTACHURL%/GGA_Kit_Reaction_Calculation_Spreadsheet.xlsx][GGA_Kit_Reaction_Calculation_Spreadsheet.xlsx]] ----------------------------------- Mix samples well by pipetting, then run the reaction on the thermocycler under the following conditions: | *Step* | *Temperature* | *Time* | | 1 | 42°C | 1 min | | 2 | 16°C | 1 min | | Cycles 1-2: | Repeat 30x | | | 3 | 60°C | 5 min | * Transform 2 μL assembly reaction and plate recovery on LB + Selective Antibiotic [[GoldenGateAssemblyProtocolsMainPage][Back to Golden Gate Protocols]] ---------------------------------------------------------- -- Main.KateElston - 29 Jan 2018

   

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xlsx	GGA_Kit_Reaction_Calculation_Spreadsheet.xlsx	r1	manage	12.1 K	2021-11-03 - 20:58	KateElston

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Contributors to this topic KateElston, SeanLeonard, PatrickLariviere, VictorLi

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