[[http://barricklab.org/twiki/bin/view/Lab/GoldenGateAssemblyProtocolsMainPage Back to Golden Gate Protocols]] ---+ First-Stage Plasmid (Transcriptional Unit) Assembly Once you have all your desired part plasmids built you can assemble them into Transcriptional units (TU). Typically these plasmids will be assembled with part plasmids for promoter + RBS, coding sequence, terminator, and TU specific connectors; variations on this basic strategy are easily managed though as long as the requisite overhangs are present ([[http://barricklab.org/twiki/bin/view/Lab/ProtocolsBTKDesignANewPart Described here]]). ---+++ Assembly reaction Total volume will be 20 μL; you will need 10 fmol of each part plasmid for this assembly. Typically the pYTK095 part plasmid (ColE1 origin, sfGFP dropout) is used as the backbone for this assembly. If toxicity issues may be a factor, lower copy number vectors can easily be used in its place. * 10 fmol of each part plasmid * 2 μL of 10× T4 DNA ligase buffer (Promega) * 1 μL of BsaI-HFv2 ( *DO NOT* use BsaI-HF*) * 1 μL of T4 DNA ligase (Promega) * x μL water up to 20 μL total. *BsaI-HF is optimized for different buffer conditions and doesn't work well ** Promega T4 Ligase buffer has been shown to offer the highest activity for BsaI enzyme. Other T4 ligase enzymes will work, but Promega enzyme with Promega buffer is highest efficiency. ------------------------------------------------ Mix samples well by pipetting, then run the reaction on the thermocycler under the following conditions: | *Step* | *Temperature* | *Time* | | 1 | 37°C | 1.5 min | | 2 | 16°C | 3 min | | Cycles 1-2: | Repeat 25x | | | 3 | 37°C | 5 min | | 4 | 80°C | 10 min | * Transform 2 μL assembly reaction and plate on appropriate antibiotic Due to the nature of attempting to assemble a high number of plasmids in one reaction there are often issues that arise: [[http://barricklab.org/twiki/bin/view/Lab/ProtocolsGoldenGateTroubleshooting Check out troubleshooting tips here]] NEB has suggestions for different reaction times based on number of inserts as shown [[https://www.neb.com/protocols/2015/03/04/golden-gate-assembly-protocol-for-using-neb-golden-gate-assembly-mix-e1600][here]] NEB also found better fidelity for extreme numbers of inserts using extreme numbers of inserts using static temperatures as shown [[https://www.biorxiv.org/content/10.1101/2020.12.22.424019v1][here]] --- [[http://barricklab.org/twiki/bin/view/Lab/GoldenGateAssemblyProtocolsMainPage Back to Golden Gate Protocols]] --- -- Main.KateElston - 29 Jan 2018
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ProtocolsBTKAssembleSingleTUPlasmid
Topic revision: r11 - 2021-05-27 - VictorLi