So I posted the above meme on Twitter, and it got some good laughs.

But while I was cutting my DNA bands after my DNA digestion (please see my previous post on this, but it’s step 5 of the cloning stage), it occurred to me that I could take a photo of what DNA actually looks like for us in the lab.

Unfortunately I thought of it after I’d begun cutting out my bands, so you’ll have to excuse the broken up DNA gel.

So there’s two halves of a gel (one on top of the other). The gel itself is made up of a special buffer and 1% agarose (think of it like a refined agar, which has become very popular of late as a vegan substitute for gelatin).

The bright yellow-green bands are DNA. You can see the DNA ladder on the bottom half of the gel in the far left. That ladder is a marker that tells me how big my DNA bands are.

In this instance, we measure DNA by kilobases (kb). One base (b) is one nucleotide, whether it be adenine (A), thymine (T), guanine (G), or cytosine (C), which make up our DNA. One thousand bases = one kb.

What’s happened is that I’ve added my cut plasmid DNA (alongside a special dye that makes the DNA fluoresce, or shine brightly under this special blue light or UV) to small wells I made in the gel (see if you can spot it in my gel photos).

When I run an electrical current, the DNA, which is slightly negatively charged, moves down the gel toward the positive terminal. BUT- bigger pieces of DNA move slower than smaller ones through the agarose mesh/gel.

So, for instance, if your sample contains a piece of DNA that is 5 kb, and another that is 1 kb, the shorter fragment is going to travel faster through the agarose gel- simply because it’s smaller and can fit through the gaps in the agarose, faster. That’s why the gel shows a separation of bands corresponding to their size. The bottom fragments are smaller than the top fragments, and it cascades down according to size.

This whole process is very similar to how we separate proteins on an SDS-PAGE gel. Major differences are the samples (DNA and protein are very different things) and what the gel is made of (polyacrylamide vs agarose), but the theory (electrical current and gel matrix) is similar.

Anyway, once I’ve separated out my cut insert (~1.3 kb) from the plasmid it was in (which, when it has no insert, is roughly ~5.1 kb), I can literally cut it out of the gel with a scalpel, put it in some tubes, and purify the DNA using a special kit. Do see the previous post if you want to know more about these steps.

Just as an FYI, this particular insert is the dark blue insert I was trying to cut out of plasmid #5 to put into plasmid #2 in the Clone Woes post. I’ve fixed my problem by using a different combo of choppy choppy enzymes (BamHI and NotI instead of BamHI and EcoRI).

I think I managed to get the red insert from plasmid #4 into plasmid #2, so today I zapped my Coxiella with this newly made plasmid. I do need to sequence the plasmid still, just to make sure nothing funky has happened during the cut and paste process, but may as well just beat the cue and start trying to get it into Coxiella while I’m at it. Because I cut it out of a pre-existing and already sequenced plasmid, the chances of the insert mutating are very low, but… I’ve probably jinxed myself now. Dammit.

So many plasmids… All at different stages of cloning… Head hurts! 😦

Categories: Ph D posts

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ABugsLife

A Ph. D graduate in Microbiology, residing in Victoria, Australia. Currently working in multiple locations but still in the STEM field. 👀 🦠 🧫 🧬