![]() ![]() The earliest documented use of cells grown for scientific purposes dates back to the late nineteenth century. So, why would we want to grow immobilized rather than free cells, or vice versa? As you might imagine, there are advantages and disadvantages to culturing cells in a free or immobilized state depending on the application - and therein lies the real difference. Free cells are just that - free and able to move around while growing. At the most basic level, immobilized cells differ from free cells in that they grow while adhered to a substrate. Hello Yvonne, It sounds like you are interested in learning more about the different ways in which cells and microorganisms can be grown. You may be able to devise a cloning strategy that requires the use of every one of these DNA modifying enzymes, but you should also ask yourself why? When devising a cloning strategy, the simplest approach is oftentimes the best.įor more information on molecular cloning, please check out the following links: With cloning, as with many things in life, there exists an infinite number of ways to go about doing things. If the DNA insert lacks 5’-phosphates, it would need to be treated with PNK so as to be ligated with a CIP-treated vector. Blunt cloning strategies typically involve the use of T4 DNA ligase, and the cloning vector is often treated with CIP in order to prevent vector re-ligation. Instead, Klenow would be the enzyme of choice for generating blunt ends from DNA templates with overhangs. As we have already noted, TDT is not often used in cloning experiments and it is more likely to generate 3’ overhangs than blunt ends. Now, on to your question: could a blunt cloning strategy be devised that uses all these enzymes? Most likely, no. ![]() “CIP-ing” a vector after it is digested, but before it is used in a ligation reaction with another DNA fragment, is a very common technique used to reduce the formation of re-ligated vectors that lack an insert. Contrastingly, CIP is often used in cloning to prevent the re-ligation of a cloning vector that has complementary DNA ends - in this way, the vector will not have any free 5’-phosphates and won’t be able to re-ligate with itself. Therefore, PNK is often used to attach a 5’-phosphate to an insert that lacks a 5’-phosphate. T4 DNA ligase requires a 5’-phosphate on at least one of the two DNA fragments it joins. In contrast, calf alkaline phosphatase (CIP) removes the 5’-phosphate groups from DNA fragments. T4 polynucleotide kinase (PNK), also derived from bacteriophage T4, is used to transfer the gamma phosphate from ATP to the 5’-OH of a DNA fragment. How does T4 DNA ligase work? It forms a covalent phosphodiester bond between a 5’-phosphate group on one DNA molecule and a 3’-hydroxyl group on another DNA molecule, using ATP to carry out this task. T4 DNA ligase acts like a molecular glue that allows molecular biologists to easily insert a gene of interest into a plasmid vector. If CoCl2 is added to the reaction, TDT can often label any type of 3’ end (3’ overhangs, 5’ overhangs, and even blunt ends!).Īs its name suggests, T4 DNA ligase (derived from bacteriophage T4) ligates DNA together. While it can be used for cloning purposes, it is most commonly used for end-labeling (i.e., to attach labeled nucleotides to the 3’ end of a DNA molecule of interest). Terminal transferase, also known as TDT, is a type of DNA polymerase that adds nucleotides to the free 3’ ends of single-strand DNA. For cloning purposes, Klenow is often used to extend recessed 3’ ends of DNA fragments, something you may need to do if you wanted to ligate two DNA fragments with blunt ends (called blunt cloning). Klenow exhibits polymerase activity but lacks exonuclease activity. Klenow is a protein fragment derived from E. We’ll then address your question about how these enzymes could be used in blunt cloning experiments. Hi Girisaran, Let’s start by taking a look at your list of DNA modifying enzymes and discussing how each might be used in molecular cloning experiments. ![]()
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