New England Biolabs, M0367S, Blunt/TA Ligase Master Mix

Blunt/TA Ligase Master Mix will ligate these substrates: Related Categories DNA Ligases Applications Cloning Ligation Specification Heat Inactivation No FAQ Q: My transformations using Blunt/TA Ligase Master Mix reactions produced no colonies. What happened? A: Lack of colonies after transformation is often not indicative of a failed ligation. Competent cells vary greatly in their efficiency. The use of high efficiency competent cells can make a dramatic difference in the perceived success of a ligation. Also, the integrity of the DNA used can strongly influence the result. For example, a minute amount of exonuclease present in a DNA sample prior to ligation can chew back an overhang and prevent compatible end ligation. Additionally, salts leftover from purification steps can inhibit or decrease ligation efficiency. The DNA ends to be joined must be compatible with at least one partner containing a 5' phosphate. DNA generated from PCR needs to employ phosphorylated primers during synthesis or be treated with a kinase after synthesis. DNA prepared by restriction enzyme digestion maintains 5' phosphorylation. Blunt ends can be joined to any other blunt ends, while fragments with overhangs need to base pair correctly to be ligated. Q: Can the Blunt/TA Ligase Master Mix be used for the ligation of sticky-end fragments? A: Yes. Blunt/TA Ligase Master Mix has been optimized for the ligation of blunt-end and single-base overhangs. Sticky-end fragment ligation, a reaction that proceeds with higher efficiency, can also be accomplished with this product. Blunt/TA Ligase Master Mix improves yields for ends that typically react slowly Yields of final ligation product for all reaction conditions using high concentration T4 DNA Ligase (NEB #M0202), the Quick Ligation Kit (NEB #M2200) and Blunt/TA Master Mix (NEB #M0367). Nick, cohesive end and 3´ single-base overhang substrates were incubated for 15 minutes; the 5´ single-base overhang was incubated for 1 hour. Q: Can the ligation reaction produced by the Blunt/TA Ligase Master Mix be directly used to transform electrocompetent cells? A: No. We do not recommend the use of ligation reactions containing the Blunt/TA Ligase Master Mix for transformation of electrocompetent cells. The DNA needs to be purified prior to use in electroporation. Q: The recommended volume for a Blunt/TA 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 Blunt/TA 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 Blunt/TA 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 Blunt/TA 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 a Blunt/TA 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 Blunt/TA Ligase Master Mix. It is not necessary to use more than 5 ul in a standard transformation reaction. Q: Can I incubate a Blunt/TA Ligase Master Mix ligation reaction for longer than 15 minutes? A: Yes. However, our testing revealed that longer incubation times do not produce more transformants. Please note that we do not recommend overnight incubations because transformations of ligations incubated overnight result in a dramatic decrease in the number of transformants. Q: Can I incubate a Blunt/TA Ligase Master Mix ligation reaction at a temperature other than 25°C? A: Yes. You can incubate reactions at 16°C or 4°C. Q: My Blunt/TA 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 Blunt/TA 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 Blunt/TA 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.