{"product_id":"neb-m0369s","title":"New England Biolabs, M0369S, ElectroLigase®","description":"ElectroLigase  \u003cb\u003eRelated Categories\u003c\/b\u003e DNA Ligases  \u003cb\u003eApplications\u003c\/b\u003e Cloning Ligation  \u003cb\u003eSpecification\u003c\/b\u003e \u003cb\u003eHeat Inactivation\u003c\/b\u003e 65°C for 15 minutes  \u003cb\u003eFAQ\u003c\/b\u003e Q: My transformations using ElectroLigase® 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 left over from purification steps can inhibit or decrease ligation efficiency. Other factors affecting transformation include the requirement that 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 ligation reactions set-up with ElectroLigase® be used for transformation of chemically competent cells? A: Yes, ElectroLigase® reactions contain nothing that inhibits transformation of chemically competent cells. In fact, it works pretty well, approaching 50% of the efficiency of our Blunt\/TA Ligase Master Mix in side-by-side analyses. Q: Do I need to dilute the ElectroLigase® reaction in order to use it to transform electrocompetent cells? A: No, ElectroLigase® reactions can be used, without dilution, in electroporation procedures. Q: Can ElectroLigase® reactions be incubated for longer than 30 minutes? A: Yes, however, our testing revealed that most reactions are nearly complete by 30 minutes with some benefit seen for blunt-end ligation by going to 60 minutes. Please note that we do not recommend an overnight incubation because transformations of ligations incubated overnight with ElectroLigase® generally produce fewer transformants. Q: Can I incubate an ElectroLigase® ligation reaction at a temperature other than 25°C? A: Yes, productive ligation was observed at testing temperatures of 4°C, 16°C, 25°C and 37°C. For convenience matched to high performance, we recommend an incubation temperature of 25°C. Q: I used an ElectroLigase® reaction to transform electrocompetent cells and I experienced arcing of my sample. What happened? A: ElectroLigase reactions have been formulated to be directly compatible with electrocompetent cells. Testing on many samples does not reveal a problem using typical electroporation conditions. Arcing during electroporation can be caused by low resistance or high conductivity in the sample. The typical factors affecting conductivity during electroporation are sample temperature and salt concentration. The competent cells should be kept cold on ice and care should be taken to ensure the cuvette used does not warm up prior to being subjected to the electric field. This means wear gloves and only wipe the cuvette with a Kimwipe if necessary, taking care to not warm the sample. DNA used in ligations with ElectroLigase should be clean (e.g. column purified) and suspended in aqueous solution containing low ionic strength (10 mM Tris or less is ok). Lastly, air bubbles trapped in the sample may prevent sample contact with both electrodes of a cuvette. A few simple taps of the cuvette on a bench, after loading the DNA\/comp cell mixture, can help dislodge trapped air. ","brand":"New England Biolabs","offers":[{"title":"Default Title","offer_id":46835515752617,"sku":"M0369S","price":0.99,"currency_code":"USD","in_stock":true}],"url":"https:\/\/iright.com\/products\/neb-m0369s","provider":"Iright","version":"1.0","type":"link"}