Ready-To-Go DNA Labeling Beads (-dCTP) (#27-9240-01 GE Lifesciences) TE buffer (10 mM Tris HCl pH 7.5, 1 mM EDTA)īamHI enzyme and buffer (#R0136S New England BioLabs)ĭCTP (3000 Ci/mmol #NHG013H250UC Perkin Elmer) Hybridization cocktail (25 ml 50% dextran sulfate, 25 ml 20X SSC, 50 ml formamide, 1 ml Tris 1M, 2 ml Denhardt’s Solution 50X, 1ml 10% SDS) – prefilter before use Southern blot Step-By-Step Guideĭenhardt’s Solution 50X (5g Ficoll 70000, 5g Polyvinyl pyrrolidone, 5g BSA Fraction V in 500 ml water) DNA polymerase then utilizes the dATP32 from solution to repair these nicks and incorporates these radioactive phosphates into the backbone of the DNA. Small amounts of DNAse introduce nicks into the single stranded probe DNA. NaOH also prevents the two strands from forming hydrogen bonds due to deprotonation of all bases. What do the NaOH and the HCl do during the denaturation step?Įssentially HCl removes some/all of the purine bases from the DNA and makes the two DNA strands less sticky to each other (because there is less hydrogen bonding). It’s also very important to make sure that the glass plate on top is heavy enough so that it forces the gel and the blot together.īy using UV after the transfer step, the DNA (which has some free aldehyde groups due to depurination) can react with the nitrocellulose/nylon membrane to form covalent bonds. That’s why it is incredibly important to make sure that the blotting paper and the gel are in close contact. Through capillary action and wicking! Unlike a western blot where a voltage gradient is utilized to pull proteins into the blotting paper, in Southern Blots, the DNA merely moves over to the blotting paper overnight without much force at all. How does the DNA actually go from the gel into the blot? Even after leaving the blotting paper overnight, the transfer may not be complete! As you can imagine, the larger a piece of DNA is, the slower it will migrate through an agarose gel. It’s because these large pieces of DNA don’t transfer very well to the blotting paper. Why is there is a second denaturing step for DNA that’s 15 KB or larger? The steps for this process are described in the following image: Finally, this “probed” radioactive blot is then imaged using an autoradiograph. After the “blotting” process is complete, the DNA is then “probed” using a radioactive (or fluorescent) DNA sequence that is complementary to the sequence that you want to detect. Next, in a similar fashion to Western blots, this DNA is then transferred onto blotting paper (typically made of nitrocellulose or nylon). This will separate the DNA by size and you’ll know which well in the gel corresponds with which sample. Next, digest the DNA using DNAse and run these fragments on an agarose gel. This can be from a cell lysate or from tissue samples, etc. To execute a southern blot, first collect some DNA. As you probably understand, this is one of the most common techniques that geneticists and molecular biologists know because everything they do needs to be proved by a southern blot. You can also do some amazing things like making transgenic mice and proving that you have selected for certain genes of interest to you! All you have to do is Southern Blot any set of DNA from the mice and you’ll have concrete proof. Most commonly, you will be testing for DNA that you get from cell lysates or after creating your own plasmids. Southern Blotting is a technique that is used to detect whether you have a specific DNA sequence available in your sample. This method was originally published on here: Southern Blot Scientific Method.
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