AssemblyReactions < Lab

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---+ 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 = 650 * insert length * 50x10^-6 = *X ng*
   * 2 &mu;L of 10× T4 DNA ligase buffer (10x)
   * 1-2 &mu;L of NEB Golden Gate Assembly Mix
   * x &mu;L water up to 20 &mu;L total.

Useful spreadsheet for calculating volumes of DNA to add [[%ATTACHURL%/GGA_Kit_Reaction_Calculation_Spreadsheet0.xlsx][GGA_Kit_Reaction_Calculation_Spreadsheet0.xlsx]]

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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}%

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Topic attachments
I	Attachment	History	Action	Size	Date	Who	Comment
xlsx	GGA_Kit_Reaction_Calculation_Spreadsheet0.xlsx	r1	manage	12.1 K	2024-04-09 - 21:14	TylerDeJong

Topic revision: r3 - 2024-04-09 - TylerDeJong