New England Biolabs, M0370S, Instant Sticky-end Ligase Master Mix

Looking for additional T4 DNA Ligase formulations and variants? Related Categories DNA Ligases Applications Cloning Ligation,, Fast Cloning: Accelerate your cloning workflows with reagents from NEB Specification Heat Inactivation No FAQ Q: Do I need to transform the Instant Sticky-end Ligase Master Mix reactions immediately? A: No. The ligation/transformation efficiency does not decrease if the ligation reactions are kept at room temperature or at 4°C for a few minutes. We do not recommend incubations longer than 1 hour. If the DNA will not be used for transformation within 1 hour, we recommend storing it at -20°C. Q: Can the ligation reaction produced by the Instant Sticky-end Ligase Master Mix be directly used to transform electrocompetent cells? A: No. We do not recommend the use of ligation reactions containing the Instant Sticky-end Ligase Master Mix for transformation of electrocompetent cells. The DNA needs to be purified prior to use in electroporation. Q: 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? A: Yes. As long as the concentration of the Instant Sticky-end Ligase Master Mix in the reaction is 1X (or 50% of the total reaction volume), you can scale up the ligation reaction. Q: 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? A: We do not recommend the use of more than 5 ul of Instant Sticky-end Ligase Master Mix ligation reactions to transform 50 ul of competent cells. In some instances we have observed a decrease in the transformation efficiency when using more than 5 ul of an Instant Sticky-end Ligase Master Mix ligation reaction. DNA with compatible ends that are intact and present in the amounts specified by the protocol should be ligated by the Instant Sticky-end Ligase Master Mix. It is not necessary to use more than 5 ul in a standard transformation reaction. Q: Can the Instant Sticky-end Ligase Master Mix be used for the ligation of blunt-end or single-base overhang fragments? A: Yes, but the reaction will no longer be instantaneous. An incubation time of 15-30 minutes at room temperature is required to ensure high efficiency ligation of blunt ends or single-base overhangs. Q: My Instant Sticky-end Ligase Master Mix has frozen in my freezer. Is this a problem? A: No. We have tested the stability of the product after freezing. Free-thaw testing has confirmed that the performance after 20 freeze/thaw cycles is close to that of the original liquid mix. If the product freezes in your freezer, it's likely that the internal temperature is lower than the expected -20°C. Q: The protocol for Instant Sticky-end Ligase Master Mix indicates the reaction should not be heat inactivated. How can I inactivate the ligation activity? A: There is typically no need to inactivate Sticky-end ligation reactions. The reaction may be used directly for transformation with chemically competent cells, or the DNA can be cleaned up by ethanol precipitation, gel purification, or any suitable DNA purification column or beads to separate the ligation product from the enzyme activity and other reaction components. If you desire to halt the reaction without cleanup, you can add an equal volume of 50 mM EDTA and mix by pipetting, however, this will interfere with downstream reactions requiring magnesium or other divalent cations.