Field of Science

DNA Extraction

Todays task is extracting DNA from our T4 phages that we've spent the last two weeks growing and nurturing and caring for. To make this easier, we're using a ready-made Kit called QIAGEN (which I keep trying to pronounce qui-gon :D ). QIAGEN basically provides all the material and gives an idiot guide as to how to use it, which means that Lab Rat's task of the day involves going through said protocol and working out what is actually happening in each step.

First though, another quick notice from the Department of the Very Obvious:
  • Do not try to do DNA extractions when hung-over
I feel awful.


DNA Extraction

1.Grow the phages on agarose gel. Agar gel is a gelatenous substance from seaweed that contains both agarose and agaropectin. Agaropectin contains lots of acidic sidegroups (containings sulfur and various other things like pyruvate). Agar is usually used for gels as it's cheaper, but for DNA extraction you can't risk any of the acidic agaropectin loitering around as it stops the extraction enzymes from working. Instead you use just pure agarose.

2.Isolate phages. This is done by peeling off the top layer of agarose and using a centrifuge to spin it down to the bottom, leaving the phages in the supernatant (the liquid left behind after centrifuging). Push the supernatant through a couple of filters to remove anything else (bacteria mostly) and you're left with a pure phage solution.

3.Now the DNA extraction can start. The first instruction in the kit is 'add buffer L1'. Buffer L1 is a clever mixture of various different enzymes and buffer solutions which breaks down any bacterial RNA or DNA that might be left in your solution.

4.Next buffer L2 is added. This precipitates out the phage particles; essentially it clumps all the bacteriophage's together making them easier to extract.

5.Centrifuge to collect the phage. The centrifuge is the big fast-spinning machine that pellets all the phages down into a neat little, well, pellet. Very useful machine, and it would be even more useful if ours worked properly :(

6.Resuspend the phage in buffer L3. I suspect this is just a growth medium, to turn the phage pellet into a phage suspension.

7.Add buffer L4. Buffer L4 contains the well known SDS (which comes up very often in various extractions). SDS is a detergent which essentially breaks all the proteins down. As viruses pretty much just consist of proteins and DNA this means that the only whole thing left in the test-tube at this point is the DNA. Still quite a way to go though, so here's a diagram of a centrifuge:8. Add buffer L5, mix and centrifuge. It does not actually say, but i suspect L5 is precipitating the proteins that have just been cut up. The centrifuge will then pellet the proteins down to separate them from our DNA.

9. Equilibrate a QIAGEN-tip 20 by adding buffer QBT. This is where we hit the Magic Event Horizon (MEH) at top speed. I have no idea what is in the buffer or exactly how a QIAGEN-tip works, which is probably a good thing for copyright in general. You can get to QIAGEN to find out more about it by going here: QIAGEN

10. Allow the residue from step 8 to flow through the QIAGEN tip. One thing I do know about the tips is that they contain resin. The resin traps the DNA on it allowing the rest of the phage to wash through (essentially this will just be any liquid medium, as all the proteins have technically been removed by the centrifuge in step 8)

11. Wash the QIAGEN tip with buffer QC. This washes the DNA (which is trapped on the filter) removing any last impurities.

12. Collect DNA with buffer QC into a clean tube. The QC in some way (Magical Event Horizon fast approaching) allows the DNA to flow through the filter and into the new tube. Finally the DNA! We now (should) have a solution containing nothing but phage DNA. Just got to collect it.

13. Precipitate the DNA by adding isopropanol. Isopropanol is another old favourite, it just clumps DNA, making very very hard-to-see pellets. You've probably guessed by now but the next stage is: centrifuging, to collect the pellet.

14. Wash pellet with ethanol. Ethanol removes any residual salt as there will be some magnesium and various others in the DNA (DNA has an overall negative charge due to the phosphate backbone which collects positively charged salts). Washing involves adding ethanol, shaking very gently and then (surprise surprise) centrifuging to pellet the DNA again and remove all the ethanol.

15. Allow DNA to dry and redissolve in buffer. Buffer just keeps the DNA happy and stops it disintegrating.

And that's it! We now have a little glass bottle containing a solution of pure DNA.

Science really is just like cookery =D


John Farrell said...

Quick question: you seem to use the word 'protocol' largely to refer to what laypersons would mean by 'directions'.

But--just curious as to whether 'protocol' includes specific kinds of equipment: lab tools, depending on what task you are undertaking.

John Farrell said...

Also...about how long does this entire extraction process take. Was this the work of a single day? Two days?

Lab Rat said...

Before you do an experiment you (ideally) write out what is called a 'protocol sheet'. This is a list of everything you need to do in the right order and sometimes (e.g if there are other people needing the centrifuge) the timings as well.

What you are doing is therefore refered to as the 'experimental protocol'. I don't know if thats just an english thing or a more general scientific thing.

You will use specific equipment to carry out the protocol, but that depends on what it is. DNA extraction protocol uses very different set of equipment to a phage harvesting protocol.

Yes, I guess a protocol is just a set of directions. :)

The extraction process takes about 5-6 hours, so we do get it all done in one day (thankfully).