The Basics of Extracting DNA from Liquid Media (Old School)

Today's focus is on isolating DNA from bacteria.

I'll outline the basic steps, sans detailed numbers.

Note: This overview uses phenol and chloroform, which are both  dangerous substances. Only work with these substances if you are trained, have the proper safety gear ( gloves, lab coat, safety googles and a fume hood), emergency resources, and if there is someone else in the lab with you. (Never work alone.)

Step 1: Spin

Starting with a solution of cells in media derived from one colony, spin the cells in a centrifuge till the pellet is at the bottom of the tube.  The fluid that remains is called the supernatant. Discard it without disrupting your pellet. Don't feel guilty.

Step 2: Rip Cells Apart with a Lysis Buffer
Mix  a few milliliters (say.. 10 ml)  of Lysis Buffer:
100 mM EDTA, 
10 mM Tris (pH 7.5)
 and 1% SDS. 

Add a roughly equal amount of lysis buffer as you have bacteria. Using a pestle and vortexor, grind up your cells into they are completely lysed and stop begging for mercy. Spin this solution down and keep the supernatant.

Step 3: "Coagulate" Proteins and Start Isolating Nucleic Acids with Phenol

Now add an equal volume of phenol as your supernatant.  Mix by inversion a couple of times. Spin solution. Pipette the top layer into a new tube. Discard the rest.

Step 4: Use 1:1 Phenol Chloroform

Add 1:1 phenol chloroform in equal volume to solution. Mix again by inversion. Spin. Then pipette off top layer to keep.

Step 5: Repeat One More time, but now with 100% Chloroform
Add chloroform in equal volume solution. Mix again by inversion. Spin. Pipette off top layer to keep.

Step 6: Add 7.5 M NH4OAc

Pipette  1/2  of your  solution volume of 7.5 M NH4OAc to your solution.

Step 7: Add ethanol

Add enough ethanol to reach a 66% ethanol solution. Invert a few times.

Step 8: Wait patiently

Wait about 10 minutes with your solution at room temperature (It is recommended for steps that you do things on ice to prevent degrading DNA accept for when re-suspending your DNA pellet at the end.) Twiddle your thumbs.

Step 9: Spin Spin Spin

Spin sample until your DNA pellet is on the side of the tube. Decant ethanol without disrupting or moving your pellet.

Step 10: Wash with 70% ethanol.

Add 70% ethanol. Centrifuge and then decant supernatant carefully without harming your pellet.

Step 11: Dry and Wet Again

Let ethanol evaporate off the tube.  Then re-suspend DNA in 20-100 ul of TE buffer or distilled water lab grade water. Give the DNA time to dissolve into solution.





Once the DNA is isolated, check the concentration using a Nanodrop. A Nanodrop is a spectrophotometer that can measure the concentration of DNA, protein, or RNA, using a beam of light and Beer's law. In essence, the Nanodrop measures the absorbance of light caused by a small droplet of your solution. Then it uses an equation to calculate the concentration of your substance based on how much light got absorbed by the particulates in your droplet.


Resource and For Further Reading:  http://palumbi.stanford.edu/SimpleFoolsMaster.pdf

How to Make Starting Lab Work Easier: Part 1

I'm chronicling my adventures in the lab to bring what it's like to work as a new scientist to the forefront. 

On pipettes

 You are  given a long pointy instrument that measures to the precision of microliters. You are told to transfer tiny rain droplets of what looks like spit. Your hands have the tendency to shake. The gloves on your hands feel weird like there is less hold or contact.

The naïve biotech wannabe must first face the task of learning to pipette.  In brief, a pipette is a glorified turkey baster. During the course of a lab day,  one has to choose between a p10, p20, p200, and p1000. The numbers signify the largest amount of microliters that pipette can handle. I don't know what the "p" stands for. Probably, pipette.

Pipettes are good at sucking and spitting out. I know that's not flattering, but it will be your constant companion. 

So, you have to treat it right, but that can be a challenge. There's a lot to mess up like: 

1. Touching the tip of the pipette to any surface in the lab- That's how you get contamination in your samples.
2. Holding the pipette horizontally- Keep the pipette tip pointed down. Gravity rules all. Your pipetted material will flow up into the pipette. Then, it will be dirty the next time someone else uses it.  I'm no clean freak. I don't take messiness seriously, except for in the lab. In that environment, being a little unsanitary could mean a project delay of months and wasted money (thousands). What if someone's working on a cure for HIV and you delay that? Sad. It could also cause a biohazard.
3.  Get a clean pipette tip every time you pipette into a new container. Don't double dip.
4.  When ejecting a pipette tip, (you push a fancy ejection button) don't put it inside the ejection container. Keep it a few inches above. It's cleaner that way.

Basic Use: MEASURE, STAB, SUCTION, EXPULSION

Slide a rotating knob to measure your amount in microliters.

Stab pipette into a matching sized tip.

Press plunger down, and stop when you first meet resistance.

Place in the  sample and remove thumb pressure from the plunger.

Release your sample by pressing the plunger down once again to the second click. 

Getting used to the feeling of when you're pipetting to the first or second click sometimes takes time. You need it for accuracy, though.

Quick tips:

Practice pipetting droplets of water on wax paper until you get a feel for the instrument.

Pipette your cheap liquids first. It is ideal to start with something like water  and to destroy a water only sample than to start with an expensive enzyme and mess that sample up.

If the material you are working with is viscous, dip the pipette tip only near the surface. This prevents extra material from sticking to the outside of the pipette.

Try to place the sample you are transferring into the bottom of the tube. 

Avoid blowing bubbles.

Shaking hands can get better with increased comfort and the loss of newbie's nerves .  You can stabilize your arms further by putting both elbows on a table and holding your wrist with your nonpipetting hand.

Always think a second ahead about what container you need to open just before you pipette up liquid. Close containers shortly after to minimize contamination.