The Role of GTE in DNA Extraction

The Role of GTE in DNA Extraction
••• gorodenkoff/iStock/GettyImages

In molecular biology, good buffer choice and preparation for different DNA isolation steps can be the difference between moving on to protein expression or opening up another maxi-prep kit to start over. In spite of their crucial importance, buffer recipes are often just that–recipes written without explanation or rationale for the different components. One such buffer is glucose-tris-EDTA or GTE buffer.

What is GTE Used For?

GTE is used to resuspend bacterial cell pellets prior to lysing (breaking open) the cells and harvesting the plasmid DNA inside. Lysozyme, which softens the cell membranes, is often added along with the GTE buffer. Achieving a homogenous suspension of whole cells during this step so that the subsequently added lysis solution can get to all of the cells is key to getting good DNA yields. GTE is designed to do this while also providing a stable environment for the DNA.

Glucose for Osmolarity

50mM (millimolar) glucose sugar is added to GTE buffer to maintain osmolarity where the solute concentration outside the cells is close to that inside the cells. This prevents premature cell lysis, which can cause lower DNA yields to due to aggregation and degradation. The other components of the buffer also contribute to the osmolarity of the solution, but glucose, being a non-electrolyte, is a good choice because it does not interfere with the solution's buffer properties.

Tris for pH Stability

Tris is the short name for tris(hydroxymethyl)aminomethane, which is a very common pH buffer. In the case of GTE buffer, the acid salt (Tris-HCl) is added to the buffer at a concentration of 25mM. This maintains the pH of the solution at a near-physiological 8.0, an ideal pH for preventing acid hydrolysis (degradation) of the plasmid DNA and unwanted side reactions of the other cell components.

EDTA Prevents DNA Degradation

EDTA, or ethylenediaminetetraacetic acid, captures or "chelates" metal ions out of solution, preventing them from participating in unwanted side reactions. In GTE buffer, EDTA is added at 10mM. Its primary purpose is in the buffer to round up free zinc, magnesium, and calcium, thereby preventing DNA degradation by certain pathways that require those metals.

Some Important Tips

Keep your GTE buffer cold and make it in small quantities to prevent the growth of unintended bacteria in it. Sugar and the controlled pH make for a great growth medium. Always use purified water. Tap water can have an excess of metal ions from the pipes, which can overwhelm the ability of the EDTA to capture. If you suspect the presence of interfering RNA in your sample, add RNase A at 100 micrograms per milliliter to your GTE buffer to eliminate the problem.

Related Articles

How to Calculate RNA Concentration
Components of Lysis Buffers
What Are the Effects of an Alkaline pH on the Structure...
What Does Ethanol Do in a DNA Extraction?
How a Sample of DNA Is Collected and Prepared for Study
Why Is Sodium Used in DNA Extraction?
How to Calculate RNA Concentration
What Is the Function of Tracking Dye in Gel Electrophoresis?
How to Dissolve EDTA in Water
The Purpose of the Buffer in Electrophoresis
How Is DNA Visualized Using Gel Electrophoresis?
How to Design an Experiment to Test How pH Affects...
What Are Biological Buffers?
The Difference Between Sucralose & Fructose
How to Isolate MRNA From a Cell
How to Make an EDTA Solution
How to Make a Citrate Buffer
Steps of DNA Transcription
Chemicals Used in DNA Analysis
How to Prepare Buffer Solutions

Dont Go!

We Have More Great Sciencing Articles!