Gibson Assembly^® Cloning Kit

The following reagents are supplied with this product:

NEB #	Component Name	Component #	Stored at (°C)	Amount	Concentration
E5510S     Multi-temperature       NEBuilder® Positive Control N2611AVIAL -20 1 x 0.05 ml Not Applicable   NEB® 5-alpha Competent E. coli (High Efficiency) C2987HVIAL -80 10 x 0.05 ml Not Applicable   pUC19 Vector N3041AVIAL -20 1 x 0.025 ml 50 pg/µl   SOC Outgrowth Medium B9020SVIAL 4 1 x 25 ml Not Applicable   Gibson Assembly Master Mix M5510AVIAL -20 1 x 0.1 ml 2 X

Features

• Assembly and transformation in just under two hours
• Flexible sequence design (scar-less cloning)
• No PCR clean-up step required
• High transformation efficiencies for inserts up to 20 kb
• Easily adapted for multiple DNA manipulations, including site-directed mutagenesis
• Includes competent cells

Companion Products

• Gibson Assembly® Master Mix
• NEB® 10-beta Competent E. coli (High Efficiency)
• NEB® 10-beta Electrocompetent E. coli
• NEB® 5-alpha Competent E. coli (High Efficiency)
• Q5® High-Fidelity 2X Master Mix
• Q5® High-Fidelity DNA Polymerase
• Q5® Hot Start High-Fidelity DNA Polymerase
• Q5® Hot Start High-Fidelity 2X Master Mix
• SOC Outgrowth Medium
• Monarch® Plasmid Miniprep Kit 
• Monarch® DNA Gel Extraction Kit
• Monarch® PCR & DNA Cleanup Kit (5 μg)

1. We highly recommend using our web tool, NEBuilder™ to design PCR primers with overlapping sequences between the adjacent DNA fragments and for their assembly into a cloning vector.
   
   
2. Storage Note:
   The kit is shipped on dry ice. Upon arrival, store kit at -80°C. After first use, store the kit components at indicated temperatures.
   
   
3. Usage notes:
   
   To ensure the successful assembly and subsequent transformation of assembled DNAs, NEB recommends the following:
   
   
   • DNA: PCR product purification is not necessary if the total volume of all PCR products in the Gibson Assembly reaction is 20% or less of the Gibson Assembly reaction volume. Higher volumes of PCR products may reduce the efficiency of Gibson Assembly and transformation due to the elevated carryover amounts of PCR reaction buffer and unused primers present in the PCR product. Column purification of PCR products may increase the efficiency of both Gibson Assembly and transformation by 2–10 fold and is highly recommended when performing assemblies of three or more PCR fragments or assembling longer than 5 kb fragments. Purified DNA for assembly can be dissolved in ddH₂O (Milli-Q® water or equivalent is preferable), TE or other dilution buffers.
   • Insert: When directly assembling fragments into a cloning vector, the concentration of assembly fragments should be at least 2–3 times higher than the concentration of vector. For assembly of multiple fragments into a vector, we recommend using equimolar ratio of fragments.
   • Transformation: NEB 5-alpha Competent E. coli (High Efficiency, NEB #C2987) provided with the kit are recommended for use for assembled products of less than 20 kb in size. It is also possible to use other NEB competent E. coli strains, with the exception of BL21, BL21(DE3), Lemo21(DE3) and Nico21(DE3). For example, Shuffle T7 Express Competent E. coli can be used for the expression of a difficult to express protein. When using competent E. coli from a vendor other than NEB, we have seen decreased robustness of transformation with the Gibson Assembly reaction.
   • Electroporation: Electroporation can increase transformation efficiency by several logs. When using the Gibson Assembly Master Mix product for electroporation, it is necessary to dilute the reaction 3-fold and use 1 μl for transformation. Should you require the use of Electrocompetent cells, please use the Electrocompetent Cells Transformation Protocol.

1. Gibson, D.G. et.al. (2009). Nature Methods. 343-345.
2. Gibson, D.G. et al. (2010). Nature Methods. 901-903.
3. Barnes, W.M. (1994). Proc. Natl. Acad. Sci.. 91, 2216-220.

1. Gibson Assembly® Protocol (E5510)
2. Gibson Assembly® Chemical Transformation Protocol (E5510)
3. Gibson Assembly® Electrocompetent Cells Transformation Protocol (E5510)

• Synthetic Biology/DNA Assembly Selection Chart

• NEBuilder® Assembly Tool

1. I am not sure whether to choose NEBuilder HiFi DNA Assembly or NEB Gibson Assembly? How are the products different?
2. What are the advantages of this method compared to traditional cloning methods?
3. How large of a DNA fragment can I assemble?
4. How many fragments of DNA can be assembled in one reaction?
5. What are the shortest overlaps that can be used with this assembly method?
6. What are the longest overlaps that can be used with this method?
7. Can ≤ 200 bp dsDNA fragments be assembled by this method?
8. Can ssDNA oligonucleotides be combined and assembled with dsDNA fragments?
9. Will the reaction work at other temperatures?
10. Is it necessary to purify PCR products when doing DNA assemblies using either NEBuilder HiFi DNA Assembly Master Mix or Gibson Assembly Master Mix?
11. Is it necessary to inactivate restriction enzymes after vector digestion?
12. I would like to produce overlapping dsDNA fragments by PCR. Do I need to use PCR primers that have been purified by PAGE or HPLC?
13. Can I use a 15-nt overlap that is entirely composed of His-tag repeats (i.e., CACCACCACCACCAC)?
14. I would like to assemble ssDNA oligonucleotides into dsDNA fragments. Do I need to use oligonucleotides that have been purified by PAGE or HPLC?
15. Can I PCR-amplify the assembled product?
16. What should I do if my assembly reaction yields no colonies, a small number of colonies, or clones with the incorrect insert size following transformation into E. coli?
17. How can I reduce the number of vector-only background colonies?
18. What type of competent cells are suitable for transformation of DNA constructs created using Gibson Assembly?
19. Can I use electroporation instead of chemical transformation?
20. Are there any differences between the Gibson Assembly Master Mix (NEB #E2611) and Gibson Assembly Master Mix included in the Gibson Assembly Cloning Kit (NEB #E5510)?
21. Are there any differences between the requirements for 2-3 fragmentassemblies versus 4–6?
22. The Gibson Assembly Master Mix control reaction is not giving me any colonies. Why?
23. When using a polymerase that doesn't contain a 3'-5' exonuclease activity (such as Taq DNA Polymerase) to amplify fragments to be used in a Gibson Assembly reaction, should I be concerned about the potential 3' mismatch generated by the addition of a non-templated nucleotide?
24. When my Gibson Assembly Cloning Kit arrived, it was stored at -80°C. Will this harm the Gibson Assembly Master Mix?
25. I would like to use NEBuilder but am concerned about user data privacy. How does NEB handle the information that I enter into NEBuilder?
26. Can I Use other primer design tools such as SnapGene for Gibson Assembly, to design primers for NEBuilder HiFi DNA Assembly?
27. What antibiotic resistance is encoded in the NEBuilder Positive Control (assembly control)?

• Gutjahr A, Xu SY (2014) Engineering nicking enzymes that preferentially nick 5-methylcytosine-modified DNA Nucleic Acids Res; 42(9), e77. PubMedID: 24609382, DOI: 10.1093/nar/gku192

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

• E5510S_v1
• E5510S_v2

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

• E5510S_v1_10010007
• E5510S_v1_10011744
• E5510S_v1_10014000
• E5510S_v1_10016306
• E5510S_v1_10020252
• E5510S_v1_10023179
• E5510S_v1_10025755
• E5510S_v1_10028551
• E5510S_v1_10032180
• E5510S_v1_10035480
• E5510S_v1_10037106
• E5510S_v1_10039410
• E5510S_v1_10042672
• E5510S_v1_10047060
• E5510S_v1_10050180
• E5510S_v1_10054389
• E5510S_v1_10057764
• E5510S_v1_10058461
• E5510S_v1_10060048
• E5510S_v1_10064424
• E5510S_v1_10068993
• E5510S_v1_10071379
• E5510S_v1_10078121
• E5510S_v1_10082114
• E5510S_v1_10085844
• E5510S_v1_10090950
• E5510S_v1_10095368
• E5510S_v1_10103373
• E5510S_v1_10108177
• E5510S_v1_10114329
• E5510S_v1_10116431
• E5510S_v1_10117474
• E5510S_v1_10120010
• E5510S_v1_10125855
• E5510S_v1_10131381
• E5510S_v1_10135797
• E5510S_v1_10141628
• E5510S_v1_10147015
• E5510S_v1_10152264
• E5510S_v1_10155360
• E5510S_v1_10159785
• E5510S_v1_10164449
• E5510S_v1_10173933
• E5510S_v1_10176841
• E5510S_v1_10168569
• E5510S_v1_10180824
• E5510S_v1_10184924
• E5510S_v1_10187117
• E5510S_v1_10196227
• E5510S_v1_10201888
• E5510S_v1_10205781
• E5510S_v1_10210308
• E5510S_v1_10218635
• E5510S_v2_10230125
• E5510S_v2_10236679
• E5510S_v2_10241698
• E5510S_v2_10246019
• E5510S_v2_10252446
• E5510S_v2_10254318

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

• NEBuilder^® Positive Control
• NEB^® 5-alpha Competent E. coli (High Efficiency)
• pUC19 Vector
• SOC Outgrowth Medium
• Gibson Assembly Master Mix

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

Licenses

Limited Warranty: The Gibson Assembly® Master Mix and Gibson Assembly Cloning Kit are warranted to perform according to specifications stated on the certificate of analysis. No other warranty is made, whether express or implied, including any warranty of merchant ability or fitness for a particular purpose. This warranty limits NEB’s and its licensors’ liability to only the price of the product. Neither NEB nor its licensors shall have any responsibility or liability for any special, incidental, indirect or consequential loss or damage whatsoever.