TITLE: No Title
AUTHORS: 

[DESCRIPTION]
Taken directly from NEB's website, this is the protocol for using their optimized sticky-end ligation and transformation. For more information, see <a href="https://www.neb.com/products/m0370-instant-sticky-end-ligase-master-mix" target="_blank">https://www.neb.com/products/m0370-instant-sticky-end-ligase-master-mix</a>.<br /><br />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 <em>E. coli</em> without dilution. <br /> <br /> * 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.
<h4>Product Source</h4>
Purified from an <em>E. coli </em>strain containing a recombinant gene encoding T4 DNA Ligase.
<h4>Reaction Volume Definition</h4>
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. <span class="bodycopy">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.<br /><br /></span><span class="bodycopy">Chemically competent strains of <em>E. coli</em> (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.<br /><br /></span><span class="bodycopy">Transformation efficiencies around 2 x 10<sup>6</sup> cfu/µg are typically achieved for recombinant cohesive end substrates (vector + insert), using cells with a 7 x 10<sup>8</sup> 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.).</span>

[STEPS]
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?. 
?.