---+ Assembly Reactions *Under Construction* Protocols from NEB [[https://www.neb.com/protocols/2018/10/02/golden-gate-assembly-protocol-for-using-neb-golden-gate-assembly-mix-e1601][BsaI]] and [[https://www.neb.com/protocols/2020/01/15/golden-gate-assembly-protocol-for-using-neb-golden-gate-assembly-kit-bsmbi-v2-neb-e1602][BsmBI]] toolkits All parts to be assembled must have correct recognition sites for BsaI or BsmBI, and correct 4bp overhangs to assemble in order. General Golden Gate Assembly protocol utilizing NEB Golden Gate toolkits (BsaI or BsmBI) In a PCR tube add: * 50 fmol of each assembly part * 2 μL of 10× T4 DNA ligase buffer (10x) * 1-2 μL of NEB Golden Gate Assembly Mix * x μL water up to 20 μL total. ----------------------------------- Mix samples well by pipetting, then run the reaction on the thermocycler under the following conditions: For BsaI run at 37°C, for BsmBI use 42°C | *Step* | *Temperature* | *Time* | | 1 | 37°C/42°C | 1.5 min | | 2 | 16°C | 3 min | | Cycles 1-2: | Repeat 30x | | | 3 | 60°C | 5 min | *Notes* If assembling insert into backbone, use 2:1 ratio of insert to backbone Reaction volumes can be cut in half to save reagents NEB suggests various thermocycler protocols depending on the complexity of your assembly as listed on their protocol pages, for extreme cases reaction can be run for up to 90 cycles. -- %USERSIG{VictorLi - 2021-11-03}% ---++ Comments %COMMENT%
This topic: Lab
>
WebHome
>
ProtocolList
>
GoldenGateAssemblyProtocolsMainPage
>
GoldenGateAssembly
>
AssemblyReactions
Topic revision: r1 - 2021-11-03 - VictorLi