Barrick Lab :: ProtocolsBTKAssembleMultipleTUPlasmid

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---+ 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 &mu;L; 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 &mu;L of NEB 10× T4 DNA ligase buffer
   * 1 &mu;L of NEB Golden Gate Enzyme Mix BsmBI-v2
   * x &mu;L water up to 20 &mu;L total.

For easy part volume calculation: [[%ATTACHURL%/GGA_Kit_Reaction_Calculation_Spreadsheet.xlsx][GGA_Kit_Reaction_Calculation_Spreadsheet.xlsx]]

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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 &mu;L assembly reaction and plate recovery on LB + Selective Antibiotic

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-- Main.KateElston - 29 Jan 2018

Attachments

Topic attachments
I	Attachment	History	Action	Size	Date	Who	Comment
xlsx	GGA_Kit_Reaction_Calculation_Spreadsheet.xlsx	r1	manage	12.1 K	2021-11-03 - 20:58	KateElston

This topic: Lab > BroadHostRangeToolkit > ProtocolsBTKAssembleMultipleTUPlasmid
Topic revision: r9 - 2021-11-03 - KateElston