DNA / RNA Manipulation and Analysis

Cloning Large or Complex DNA Fragments

Sometimes you know a project is going to be a pain before you even start it. For me, that is whenever I need to clone large (> 3 kb) or complex (e.g., a sequence with repeats) DNA fragments. Long and complex DNA fragments are more likely to create challenges during cloning. Such projects require extra care in just about…

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How DNA Extraction Is Different in Plants

I know what you are thinking, everything is made of cells, so how different can DNA extractions be in plants? The answer is… sort-of different. The overall concept is the same. Cell membranes are lysed, DNA is separated from other cell materials, washed a few times, and then resuspended in water or Tris-EDTA buffer (aka…

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The Three Ts of Introducing Foreign DNA: Transfection, Transduction, and Transformation

Introducing foreign DNA into bacterial or eukaryotic cells is a common molecular biology technique. Some of the terminology involved can be confusing: transformation, transfection and transduction. What do these words actually mean? To make matters worse, sometimes people use these terms interchangeably, resulting in confusion between colleagues and collaborating labs. Here then is your definitive guide…

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AAV Production Part II: Virus Purification

In Part I of AAV Production, I described how to produce crude (non-purified) AAV. In this article, I am going to tell you how to purify that crude prep. Virus purification is usually done by gradient ultracentrifugation. Two common methods involve gradients made from increasing concentrations of cesium chloride or iodixanol. A cesium chloride prep…

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