![]() ![]() Standard, Restriction Enzyme-based cloning What plasmid should you put your gene(s) of interest into? Read about origin of replication, copy number, and plasmid compatibility here.coli strains here, to choose the best one for your application. What organism is your host, and what is the genotype? Learn about all sorts of E.There is a 30 day free trial with any valid e-mail address.Īlso, if you are preparing your own electrocompetent cells for transformation, please see Bucci lab methods for a protocol. I tried a few free software tools, but after using the free trial of SnapGene, there was no going back. Maybe I can help you answer some of the questions you might have in your head when considering a cloning experiment. Instead, let's consider these things one at a time. What is the copy number of your backbone? Will that work for your desired application? Does your vector backbone have a Multiple cloning site (MCS)? Do you have the enzymes necessary for that MCS? Does your insert have any of the restriction sites you want to use for the 5' and 3' ends? Do you even want to do standard restriction cloning? I could go on and on. Design your constructs and really scrutinize all of the decisions you are making. Think about one step at a time and really be detail oriented. All of the methods and protocols that you need are out there, as long as you have an internet connection. My suggestion for learning to clone: just learn as you go. Most often, I find that I am reading the protocols as provided by the manufacturer of whatever particular reagent(s) I am using, which is almost always New England Biolabs (NEB). I see that Addgene has a protocols page, but I'll say with honesty that I haven't read these protocols. I started in a small lab with nobody who understood how to clone, and so all of my techniques I've learned from the internet (or books, but even, Molecular Cloning - Green and Sambrook, has limitations regarding depth and specificity for particular applications, though this is an essential text to be in any laboratory where cloning is conducted). 2 Standard, Restriction Enzyme-based cloningįirst, I'd like to say that I am largely self-taught in cloning methods. ![]()
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