High Complexity Golden Gate Assembly Standards Kit (24 fragment lac cassette)
(Kit #
1000000221
)
Depositing Lab: Eric Cantor
A positive control for high-complexity golden gate assembly. Junction sites were predicted using comprehensive end-joining ligation fidelity and bias data to allow high accuracy assembly.
This kit consists of one 96-well plate, and will be shipped as bacterial glycerol stocks on dry ice. Samples should be frozen at -80 °C immediately upon arrival. Individual plasmids can be ordered from each plasmid page and will be shipped as bacterial stabs.Original Publication
Comprehensive Profiling of Four Base Overhang Ligation Fidelity by T4 DNA Ligase and Application to DNA Assembly. Potapov V, Ong JL, Kucera RB, Langhorst BW, Bilotti K, Pryor JM, Cantor EJ, Canton B, Knight TF, Evans TC Jr, Lohman GJS. ACS Synthetic Biology. 2018 Nov 16;7(11):2665-2674. doi: 10.1021/acssynbio.8b00333. PubMed (Link opens in a new window) Article (Link opens in a new window)
Description
This set contains a destination vector pGGAselect (chlor) and 24 fragments encoding part of the lac operon in pUC57 (amp) holding plasmids. The destination vector pGGAselect can be used as a cloning vector for Golden Gate Assembly using BsaI, BsmBI, and BbsI. The 4 bp overhang ends of 24 fragments of lac cassettes assemble with high fidelity and efficiency, with a design based on the comprehensive profiling of the ligation fidelity of T4 DNA ligase. This set can be used as a positive control for a complex assembly using BsaI via blue/white screening as E.coli cells that carry the correctly assembled lac cassettes form blue colonies on an LB plate with X-gal.
- 24-fragment Golden Gate Assembly of lac cassettes.
Kit Documentation
Junction sequences of the 24 fragments of lac cassettes can be found in Tables S4 in the reference paper.
Reactions were performed as described in the Materials and Methods section of the reference paper.
For Golden Gate Assembly with BsaI, you can choose either NEBridge® Golden Gate Assembly Kit (BsaI-HF® v2) (Link opens in a new window) or NEBridge® Ligase Master Mix (Link opens in a new window) (BsaI-HF v2 needs to be purchased separately) and follow the protocols on its product page.
High complexity GGA assemblies can be designed using the guidelines in this reference: Enabling one-pot Golden Gate assemblies of unprecedented complexity using data-optimized assembly design. Pryor JM, Potapov V, Kucera RB, Bilotti K, Cantor EJ, Lohman GJS. PLoS One. 2020 Sep 2;15(9):e0238592. doi: 10.1371/journal.pone.0238592. PubMed (Link opens in a new window) Article (Link opens in a new window)
Web Tools (Link opens in a new window)to support high complexity assembly design.
Tools (Link opens in a new window)for visualization of GGA.
How to Cite this Kit
These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which they were created, and include Addgene in the Materials and Methods of your future publications.
For your Materials and Methods section:
“The High Complexity Golden Gate Assembly Standards Kit (24 fragment lac cassette) was a gift from Eric Cantor (Addgene kit #1000000221).”
For your Reference section:
Comprehensive Profiling of Four Base Overhang Ligation Fidelity by T4 DNA Ligase and Application to DNA Assembly. Potapov V, Ong JL, Kucera RB, Langhorst BW, Bilotti K, Pryor JM, Cantor EJ, Canton B, Knight TF, Evans TC Jr, Lohman GJS. ACS Synthetic Biology. 2018 Nov 16;7(11):2665-2674. doi: 10.1021/acssynbio.8b00333. PubMed (Link opens in a new window) Article (Link opens in a new window)
High Complexity Golden Gate Assembly Standards Kit (24 fragment lac cassette) - #1000000221
- Resistance Color Key
Each circle corresponds to a specific antibiotic resistance in the kit plate map wells.
- Inventory
Searchable and sortable table of all plasmids in kit. The Well column lists the plasmid well location in its plate. The Plasmid column links to a plasmid's individual web page.
- Kit Plate Map
96-well plate map for plasmid layout. Hovering over a well reveals the plasmid name, while clicking on a well opens the plasmid page.
Resistance Color Key
| Chloramphenicol | |
| Ampicillin |