Instant Sticky-end Ligase Master Mix

The following reagents are supplied with this product:

NEB #	Component Name	Component #	Stored at (°C)	Amount	Concentration
M0370S     -20       Instant Sticky-end Ligase Master Mix M0370SVIAL -20 1 x 0.25 ml 2 X
M0370L     -20       Instant Sticky-end Ligase Master Mix M0370LVIAL -20 1 x 1.25 ml 2 X

Application Features

• Vector construction
• Fragment assembly
• Library construction

Companion Products

• Monarch® Plasmid Miniprep Kit 
• Monarch® DNA Gel Extraction Kit
• Monarch® PCR & DNA Cleanup Kit (5 μg)

1. DNA: Purified DNA for ligations can be dissolved in dH₂O (Milli-Q^® water or equivalent is preferable); TE or other dilute buffers also work well. For optimum ligation, the amount of vector DNA should be 20–100 ng and the insert should be added at a 3-fold molar excess. For ligation volumes greater than 10 μl, increase the volume of Instant Sticky-end Ligase Master Mix such that it remains 50% of the reaction. Insert: vector ratios between 2 and 6 are optimal for single insertions. Ratios below 2:1 result in lower ligation efficiency. Ratios above 6:1 promote multiple inserts. If you are unsure of your DNA concentrations, perform multiple ligations with varying ratios.
2. Time and Temperature: Ligations performed using the Instant Sticky-end Ligase Master Mix are immediately ready to be used to transform E. coli. If it is necessary to incubate the reaction prior to transformation, performance is generally good between 4–25°C. Incubation can be performed for up to one hour at the chosen temperature without negatively impacting the performance of the mix. Blunt- and TA-ends can also be ligated by this mix but require a 5–15 minute incubation time to reach our internal performance standards. The "instant" ligation criteria were met with cohesive ends of 2–4 bp in length. 
3. Cells: Competent cells can vary by several logs in their competence. Perceived ligation efficiency directly correlates with the competence of the cells used for transformation. Always transform uncut vector as a control for comparison purposes. 
4. Electroporation: While electroporation can dramatically increase transformation efficiency, Instant Sticky-end Ligase Master Mix is not directly compatible with transformation by electroporation. It is necessary to reduce the PEG concentration. We recommend purification of the ligated DNA by spin column.
5. Biology: Some DNA sequences are not easy to clone. Sequences that form structures, including inverted and tandem repeats, are selected against by E. coli. Some recombinant proteins are not well tolerated by E. coli and can result in poor transformation or small colonies.

1. Ligation Protocol for Cloning with Instant Sticky-end Ligase Master Mix (M0370) 
2. Transformation Protocol (M0370)

• Tips for Maximizing Ligation Efficiencies

• Efficient Adaptor Ligation for the Preparation of dsDNA Libraries using the Blunt/TA Ligase Master Mix

• DNA Ligase Selection Chart
• Properties of DNA and RNA Ligases
• Substrate-based Ligase Selection Chart

• NEBcloner^®
• NEBioCalculator®

1. Do I need to transform the Instant Sticky-end Ligase Master Mix reactions immediately?
2. Can the ligation reaction produced by the Instant Sticky-end Ligase Master Mix be directly used to transform electrocompetent cells?
3. The recommended volume for an Instant Sticky-end Ligase Master Mix reaction is 10ul. I like to set-up my ligation reactions in a 20 ul volume. Can I scale-up the reaction?
4. I routinely use more than 5 ul of my ligation reactions to transform 50 ul aliquots of competent cells. When I do this with the Instant Sticky-end Ligase Master Mix my transformation plates have very few colonies. What do you think is the problem?
5. Can the Instant Sticky-end Ligase Master Mix be used for the ligation of blunt-end or single-base overhang fragments?
6. My Instant Sticky-end Ligase Master Mix has frozen in my freezer. Is this a problem?
7. The protocol for Instant Sticky-end Ligase Master Mix indicates thereaction should not be heat inactivated. How can I inactivate theligation activity?

• Troubleshooting Guide for Cloning
• Troubleshooting Tips for Ligation Reactions

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]

• M0370S_L_v1

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]

• M0370S_L_v1_0151710
• M0370S_L_v1_0161803
• M0370S_v1_10008066
• M0370L_v1_10008064
• M0370L_v1_10020317
• M0370L_v1_10026403
• M0370S_v1_10020318
• M0370S_v1_10035065
• M0370L_v1_10035067
• M0370L_v1_10048659
• M0370S_v1_10048657
• M0370L_v1_10058675
• M0370S_v1_10079384
• M0370L_v1_10095233
• M0370S_v1_10096785
• M0370L_v1_10109481
• M0370S_v1_10117912
• M0370S_v1_10134393
• M0370L_v1_10145046
• M0370L_v1_10164288
• M0370S_v1_10153908
• M0370L_v1_10179949
• M0370S_v1_10195895
• M0370S_v1_10206996
• M0370S_v1_10214129
• M0370L_v1_10226630
• M0370S_v1_10226653
• M0370L_v1_10245776
• M0370L_v1_10246823
• M0370S_v1_10245777
• M0370S_v1_10252856
• M0370L_v1_10252851

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

• Instant Sticky-end Ligase 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.