GEN LAB: DNA EXTRACTION
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Why does M. luteus suspension become more viscous after the addition of SDS?
SDS is a detergent. Upon adding SDS the bacteria will lyse (break open) and the liquid will become thicker due to the release of proteins and other cellular products.
Protein will denature at 60°C, while DNA denatures at 80°C. Explain why in terms of structural differences between protein and DNA.
- DNA has more H bonds.
- In order for DNA to denature the numerous H-bonds between both strands must be broken and the covalent bonding of sugar phosphate backbone (both requiring tons of energy). Protein, in the other hand, only requires damaging the quaternary and tertiary structure plus they posses less H bonds, which requires less energy for denaturation.
Explain what happens when a protein is denatured.
When a protein is stressed (heating/stirring/UV light exposed) the bonds that hold a protein in it's tertiary structure will begin to break. When these bonds break the protein starts to unfold and loses its function.
Describe and explain any color change at the aqueous-ethanol interface.
- It turned hot pink.
- The color change is due to the acidic nature of DNA.
What happens as the spooling rod is rotated in the aqueous-ethanol interface?
The DNA starts slowly attaching itself to the spooling rod as it rotates in the test tube.
Describe the appearance of the extracted DNA.
It looked like a thick web of strands of DNA on the spooling rod, the color was rusty white; it looked like wet cotton.
Describe what happens to the DNA after it has been immersed in the DNA suspension medium for 20 minutes or more.
The white DNA changed color to pink after being in the DNA suspension medium.
What does your answer to question 7 indicate about the pH of DNA?
Since it turned pink this indicates the pH of DNA is acidic.
What do your observations indicate about the nature of DNA?
- numerous H-bonds.
- acidic pH.
- stable at 60°C.
- white fibrous threads.
- 1- bacterial suspension + freeze-dried M. luteus. (save for step 9) cap the tube very tihtly and shake gently for 5min until bacteria goes into suspension.
- 2- M. luteus solution + SDS (avoid excess bubbling and rotate for 5min). suspension becomes viscous as the bacteria is lysed.
- 3- let the tube stand in a preheated hot water bath for 30 min.
- 4- prepare a solution of alkaline pH indicator and 95% ethanol; refrigerate for step 6.
- 5- let the lysate cool to RT.
- 6- lower a spooling rod into lysate in the spooling tube. add alkaline pH indicator-ethanol solution to the spooling tube. tip the tube to an angle to avoid layers mixing. allow for ethanol to flow very slowly down the aqueous layer. NOTE the change color.
- 7- hold the tube at a 45° so that the max interface surface is exposed. rotate the spooling rod in a continuos, clockwise motion. avoid touching sides of the tube. twirl until a mass of DNA has attached to the rod.
- 8- remove the spooling rod and gently immerse it in 95% ethanol for 2min. remove the rod from the ethanol and allow to dry for 5min.
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