Into the laboratory III

So, yesterday I set up the PCR reaction for all of my DNA samples. In theory, this should have given me a pure sample of one interesting gene from each: a gene that, if sequenced, would allow me to identify which organism the DNA came from.

Primers are very short strands of DNA in the PCR mastermix that control what gets replicated. In some tubes I used a primer for an insect gene, and in the others a primer for a gene involved in photosynthesis. You'd expect tube containing the insect gene primers to produce loads of replicates of that gene if there was cricket DNA in the sample, but to produce nothing if there was only plant DNA, and vice versa. So, how do you see whether DNA is present in the tiniest of plastic tubes containing the tiniest drops of transparent liquid? This is where it got cool.

Making DNA visible: the steps of gel electrophoresis
1. First I made a gel. This is a rectangular block of agarose, the sort of translucent jelly that you find in petri dishes (fun fact: it's made from seaweed). You melt it and pour it into a mould; it's liable to boil over and scald you with super-hot science goo.

2. Next, I added a special dye to all of my finished PCR tubes. It binds to the DNA and stains it dark blue.

3. I then immersed the gel in a tank of buffer, a liquid guaranteed to not react chemically with the gel or the DNA or anything else important.

4. Then came the difficult task of loading the DNA samples into the gel. Here's a picture of a finished one:

https://upload.wikimedia.org/wikipedia/commons/7/7f/Large_Gel_Electrophoresis_Chamber_with_Agarose_gel_inside_-_(1).jpg

See the line of tiny rectangles? These are wells: pockets in the gel where the DNA sample gets pipetted in. Unfortunately, because the whole thing is immersed in buffer, you have to squirt blue liquid into a pocket that is already full of clear liquid, which is quite difficult. But it's better than squirting clear liquid into clear liquid.

5. Finally, the electric current was switched on. DNA has a negative charge, so when electricity is run through the gel the DNA gets attracted to the positive end (the red end above - see that the DNA is loaded on the opposite side). It actually moves through the gel.

So what, you may ask? How does moving it help us to identify the DNA? Importantly, the gel is quite challenging for DNA to move through. There's a lot of drag. The bigger the piece of DNA, the more it's dragged back, so the less distance it travels over time. During a half-hour run, all of the differently-sized DNA molecules in each sample should have separated out into an orderly sequence, with the smallest reaching furthest to the red end and the largest stuck near the black.

This isn't all just theory: we can see it. The blue dye helps with loading, but it also glows brightly under UV light, even from very small DNA fragments. Here's a UV picture of my gel after it had finished running:

My, what a confusing (and badly-scanned) set of grey lines.

Working out what the bands mean
I had seven types of DNA sample:
- Kale (Ka)
- Spinach (Sp)
- Gut contents of the big black cricket (GutA)
- Gut contents of the small brown cricket (GutB)
- Leg from the big black cricket (LegA)
- Leg from the small brown cricket (LegB)
- Blank, with water run through PCR instead of DNA (-)

On the photo, you can see a stack of lines down the side of each set of bands. This is an appropriately-named ladder: a shop-bought mixture of DNA molecules of known sizes. If a sample band lines up with a ladder band, you can say the sample band is also, for example, 200 base pairs long. We aren't interested in length, though, just whether or not DNA is actually present. Did the primers for plant DNA pick up plant DNA in the samples of kale and spinach, and did the insect primers pick up the crickets? And most interestingly of all, did the plant primers pick up DNA in the cricket gut contents... because some of the crickets had been fed on either kale or spinach!

The series of lines is divided into four primers, each run on four types of DNA sample and a blank. I've traced over the bands so the important ones are visible, and moved the ladders around so everything can be sized.

1. A plant primer:

It found and replicated DNA in the kale and spinach samples only. Perfect!

2. An insect primer:

It found and replicated DNA in every cricket sample. Great!


Unfortunately, the other two primers didn't give very good results. I contaminated their reactions, as you can see from the bands in the blank columns, which should have been, well, blank:

I was never told which primers were which, so it's hard to tell what went wrong with any more detail. But the technician assured me that at least one of my crickets hadn't been starved before death, which was an uplifting note upon which to end the day.