Oct 17, 2020
NEB Instant Sticky-end Ligase Master Mix-CHEM 584
- Ken Christensen1,
- Harold Bien2
- 1Brigham Young University;
- 2Stony Brook University
Protocol Citation: Ken Christensen, Harold Bien 2020. NEB Instant Sticky-end Ligase Master Mix-CHEM 584. protocols.io https://dx.doi.org/10.17504/protocols.io.bninmcde
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: October 17, 2020
Last Modified: October 17, 2020
Protocol Integer ID: 43310
Abstract
Taken directly from NEB's website, this is the protocol for using their optimized sticky-end ligation and transformation. For more information, see https://www.neb.com/products/m0370-instant-sticky-end-ligase-master-mix.
Instant Sticky-end Ligase Master Mix is a ready-to-use 2X solution of T4 DNA ligase and a proprietary ligation enhancer in an optimized reaction buffer. Specifically formulated to rapidly ligate cohesive-end (2–4 bp) substrates and improve transformation, the mix simplifies reaction set-up and provides an optimized ratio of enzyme and buffer components. No thawing of the master mix is required as it maintains a liquid state during storage at -20°C* and no incubation time is necessary to achieve ligation efficiencies sufficient for successful sub-cloning of sticky-end substrates. Just add the master mix to DNA with compatible ends, mix and transform; thereby reducing the valuable time needed for routine ligations. Ligations for subcloning can be carried out in small volumes with low concentrations, allowing users to conserve precious DNA samples, and be used directly to transform many strains of chemically competent E. coli without dilution.
* Freezers vary in their actual internal temperature. Our testing demonstrates that the master mix is liquid at -20°C. Freeze-thaw testing at -70°C has confirmed that the performance after 20 freeze/thaw cycles is close to the original mix.
Product Source
Purified from an E. coli strain containing a recombinant gene encoding T4 DNA Ligase.
Reaction Volume Definition
1X Instant Sticky-end Ligase Master Mix with DNA substrates in a 10 μl reaction volume. A 10 μl reaction contains 1800 cohesive end units of T4 DNA Ligase.
Guidelines
Once, completed, ligation products can be used immediately for transformation or stored at -20C until ready to use. In-house testing has demonstrated that maximal transformation efficiency is achieved using between 20–100 ng of vector (sticky) and a corresponding 3-fold molar excess of the insert to be ligated into the vector.
Chemically competent strains of E. coli (commercially available or prepared by user) can be transformed by ligation products prepared using the Instant Sticky-end Ligase Master Mix. Electrocompetent cells are not compatible. Users of competent cells from other vendors may need to dilute ligation reactions 4-fold, prior to transformation, in order to achieve maximum transformation efficiency. Not all cells from other vendors will benefit from this additional step. The following protocol is recommended by NEB. Other protocols can be used but the volume of ligation reaction used should not exceed 5 μl reaction per 50 μl cells.
Transformation efficiencies around 2 x 106 cfu/µg are typically achieved for recombinant cohesive end substrates (vector + insert), using cells with a 7 x 108 calculated efficiency with uncut DNA. This corresponds to several hundred colonies on a plate when 100 μl of a 1 ml outgrowth is plated at a 1:5 dilution. As with all ligation and transformation protocols, many factors affect the calculated transformation efficiency, including purity and integrity of DNA ends, competence of the cells being transformed, media choices, incubation temperatures and times and biological effects (intact ORF in high-copy vector, toxic genes, etc.).
Materials
MATERIALS
Instant Sticky-end Ligase Master Mix - 50 rxnsNew England BiolabsCatalog #M0370S
STEP MATERIALS
Instant Sticky-end Ligase Master Mix - 50 rxnsNew England BiolabsCatalog #M0370S
Instant Sticky-end Ligase Master Mix - 50 rxnsNew England BiolabsCatalog #M0370S
Protocol materials
Instant Sticky-end Ligase Master Mix - 50 rxnsNew England BiolabsCatalog #M0370S
Instant Sticky-end Ligase Master Mix - 50 rxnsNew England BiolabsCatalog #M0370S
Instant Sticky-end Ligase Master Mix - 50 rxnsNew England BiolabsCatalog #M0370S
Instant Sticky-end Ligase Master Mix - 50 rxnsNew England BiolabsCatalog #M0370S
Instant Sticky-end Ligase Master Mix - 50 rxnsNew England BiolabsCatalog #M0370S
Safety warnings
Do not heat inactivate.
Heat inactivation dramatically reduces transformation efficiency.
Preparation
Preparation
Place master mix tube on ice and flick a few times to mix.
Instant Sticky-end Ligase Master Mix - 50 rxnsNew England BiolabsCatalog #M0370S
Ligation
Ligation
Combine 20–100 ng of vector with a 3-fold molar excess of insert and q.s. to 5μl with dH2O.
5 µL
Add 5 μl of Instant Sticky-end Ligase Master Mix, mix thoroughly by pipetting up and down 7-10 times, and place on ice. The sample is now ready to be used for transformation.
5 µL
Instant Sticky-end Ligase Master Mix - 50 rxnsNew England BiolabsCatalog #M0370S
Transformation into Mix & Go Competent Cells
Transformation into Mix & Go Competent Cells
5m
5m
Thaw a 50 µl aliquot of Mix & Go competent cells on ice.
Add 5 μl of the ligation reaction to the cells and mix by finger-flicking. Do not vortex the tube.
5 µL
- Incubate the tube on ice for 5 minutes. Do not mix.
00:05:00
5m
For anitbiotics other than ampicillin (e.g., kanamycin), add 200 μl of warm SOC to the tube. For ampicillin selective plasmids, no SOC addition or outgrowth is needed so you can proceed to STEP 9 immediately.
200 µL
Incubate for one hour at 37°C with rotation or shaking (200–250 rpm).
01:00:00
Spread the entire tube of cells (e.g., 50 or 250 µl) onto an appropriate antibiotic selection plate and incubate overnight at 37°C.