TITLE:  NEB Instant Sticky-end Ligase Master Mix-CHEM 584
AUTHORS: Ken Christensen, Harold Bien

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>Taken directly from NEB's website, this is the protocol for using their optimized sticky-end ligation and transformation. For more information, see </span><a href="https://www.neb.com/products/m0370-instant-sticky-end-ligase-master-mix" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.neb.com/products/m0370-instant-sticky-end-ligase-master-mix</span></a><span style = ":;">.</span></div><div class = "text-block"><span>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 </span><span style = "font-style:italic;">E. coli</span><span> without dilution. </span></div><div class = "text-block"> * 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. </div><div class = "text-block"><span> Purified from an </span><span style = "font-style:italic;">E. coli </span><span>strain containing a recombinant gene encoding T4 DNA Ligase. </span></div><div class = "text-block"> 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.</div></div>

[STEPS]
?. [Preparation]
Place master mix tube on ice and flick a few times to mix.
?. [Ligation]
Combine 20–100 ng of vector with a 3-fold molar excess of insert and q.s. to 5μl with dH2O.
5 µl
?. [Ligation]
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
?. [Transformation into Mix & Go Competent Cells]
Thaw a 50 µl aliquot of Mix & Go competent cells on ice.
?. [Transformation into Mix & Go Competent Cells]
Add 5 μl of the ligation reaction to the cells and mix by finger-flicking. Do not vortex the tube.
5 µl
?. [Transformation into Mix & Go Competent Cells]
Incubate the tube on ice for 5 minutes. Do not mix.
?. [Transformation into Mix & Go Competent Cells]
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
?. [Transformation into Mix & Go Competent Cells]
Incubate for one hour at 37°C with rotation or shaking (200–250 rpm).
?. [Transformation into Mix & Go Competent Cells]
Spread the entire tube of cells (e.g., 50 or 250 µl) onto an appropriate antibiotic selection plate and incubate overnight at 37°C.