EZ vision sample loading dye (not a paid endorsement, I just find it helpful)
One of my favorite under-known tools in the lab is EZ Vision sample loading buffer (not a paid endorsement, I’m just a fan!). This is a sample loading buffer for your DNA gels that has a fluorescent DNA-binding dye in it so you don’t need to put stain in the gel or stain your gel afterwards. Here’s more about how it works as well as how some other fluorescent nucleic acid stains work… (text old, video new)
blog: http://bit.ly/fluorescentstains
more on other fluorescent stains: • Fluorescent nucleic acid stains & flu...
“Yesterday” I wanted to look at small amounts of DNA - so I went for the gold! SYBR gold, that is. It’s a fluorescent nucleic acid stain, meaning that it binds to nucleic acids (RNA & DNA) and gives off light at a specific wavelength when you shine light of a different specific wavelength at it. Other fluorescent nucleic acid stains include the rest of the proprietary SYBR family (e.g. SYBR Green, SYBR Safe) as well as the non-proprietary Ethidium Bromide (EtBr) and DAPI. I’ve used many of these, but was curious as to how they work and how they compare to one another, so, a while back, in addition to looking for RNA, I went looking for information! And I want to share with you some of what I learned. There are a lot of different stains out there and I’m not going to try to compare them all, but what I will do is tell you about how they work and some qualities you should look for when choosing which to be using!
First, let me make sure we’re all on the same page regarding terminology. Nucleic acids refer to DNA & RNA (there are also some different lab-made nucleic acids which I won’t get into here). Both DNA & RNA are made up of building blocks or “letters” called nucleotides, which consist of a sugar (ribose for RNA and deoxyribose for dNA) off of which sticks a phosphate-containing group (used for linking letters together into chains) and a nitrogen-containing ringy-thing called a nucleobase (“base” for short). These bases are what make different letters unique - DNA has A, G, C, & T and RNA has A, G, C, & U - and they’re important for allowing nucleic acids to form specific base pairs to other nucleic acids (A:T or A:U) and (G:C). This specific base pairing allows for DNA & RNA to store genetic information and be copied and stuff, but it’s not that relevant for our discussion today because a good fluorescent stain shouldn’t care about what letters are present - we want a stain that stains any sequence!
One consequence of base pairing that does matter for our discussion, however, is that it allows single-stranded (ss) nucleic acids (ssRNA & ssDNA) to bind to strands of complementary nucleic acids to form double-stranded (ds) helical structures (dsRNA & dsDNA) in which the bases are “stacked” in the center like rungs on a ladder. note: for DNA, this typically happens between 2 separate DNA strands, but for RNA, which is more commonly single-stranded, regions of the same strand of RNA often fold back upon themselves to form double-stranded regions, such as hairpins, & stem-loop structures, within a single strand. Why am I telling you all this? Because the stranded-ness can affect how well some stains work. So now let’s talk about how these stains work.
There are 3 things we need in our stain. They need to be able to:
1. bind what we want them to bind (have high affinity for DNA and/or RNA)
3. not bind what we don’t want them to bind (be selective)
4. show us where they’re bound (this is where the fluorescence comes in)
Different dyes bind slightly differently, but there are 2 major modes of binding
1. intercalation (wedging in between the bases in the helix ladder, or between stacked bases in other “shapes”)
2. groove binding (binding on the “outside” in the helix’s grooves)
there are also other modes of binding, and some dyes bind multiple ways and/or unknown ways and I’ve gone down way too many rabbit holes to try to find out! So I’m going to focus on these 2.
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