Sunday, March 19, 2017

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:

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