DNA GLOW Lab A new way to investigate DNA structure V11 June 2018 Copyright by Amplyus LLC all rights reserved We bring the study of DNA structure to your hands Patent pending DNA GLOW Lab ID: 770639
Download Presentation The PPT/PDF document "DNA GLOW Lab A new way to investigate DN..." is the property of its rightful owner. Permission is granted to download and print the materials on this web site for personal, non-commercial use only, and to display it on your personal computer provided you do not modify the materials and that you retain all copyright notices contained in the materials. By downloading content from our website, you accept the terms of this agreement.
DNA GLOW Lab A new way to investigate DNA structure. V1.1: June 2018 © Copyright by Amplyus LLC, all rights reserved
We bring the study of DNA structure to your hands Patent pending
DNA GLOW Lab Review DNA structure and chemical properties. Predict how DNA will respond to changes in its chemical and physical environment. Test predictions using a fluorescent dye that allows us to visually verify aspects of DNA structure. Goals for today:
The DNA model In 1952, 23-year-old James Watson and 37-year-old PhD student Francis Crick built the first accurate model of DNA. They based their model on several sources. Notably:Data from Erwin Chargaff suggested that nucleotides were bound in pairs, adenine to thymine and guanine to cytosine. Photo51, an X-Ray diffraction image made by Raymond Gosling under the direction of Rosalind Franklin, suggested that DNA was a double helix.“This structure has novel features which are of considerable biological interest.” - A Structure for Deoxyribose Nucleic Acid Watson J. and Crick F Nature 171 , 737 (1953)
The DNA model Today, the double helix is an iconic image in science that we are all familiar with. Like all molecules, however, its structure had to be deduced using chemical tests. We now know that DNA is made of two backbones comprised of alternating five-carbon sugars and phosphate groups wrapped around nitrogenous bases which are joined by hydrogen bonds. This proposed structure leads to predictions about the molecule’s chemical properties that we can test in the laboratory.
Purines and pyrimidines Purines (adenine and guanine) have a double ring structure. Pyrimidines (thymine and cytosine) have a single ring structure. Guanine and cytosine can make three bonds. Adenine and thymine can make two bonds. The polarity of the bonds between the homologous pairs is reversed.
Base pairing A single ring pyrimidine will always bind to a double ring purine giving DNA its constant width. Hydrogen bonds hold the two strands together.
Base pairing Hydrogen bonds are weaker than covalent bonds and are more subject to changes in physical and chemical properties such as heat and pH.
Base pairing When hydrogen bonds are heated, they will break, and DNA will denature. When cooled, they will reform, and DNA will anneal.
DNA GLOW Lab: Exploring DNA Structure DNA is mixed with a dye that fluoresces only when bound to double stranded DNA. When physical or chemical conditions are sufficiently changed, DNA will become denatured and will no longer fluoresce.When conditions are returned, DNA will anneal and fluorescence will return.
Equipment needed Blue light illuminator Heat source & Or Or
Determining GC content Can you determine which sample has the highest GC content? The lowest?
Determining GC content Quickly transfer to blue light illuminator. Wait 1 minute. Heat to 95 ˚. *Continue viewing for about one minute.
Programming the miniPCR Choose +NEW Choose Create Heat Block Protocol Choose Library
Programming the miniPCR Give it an awesome and creative name Choose 95 ˚ Save and run
Determining GC content Quickly transfer to blue light illuminator. Wait 1 minute. Heat to 95 ˚. *Continue viewing for about one minute.
Determining GC content Which sample had the highest GC content? The lowest?Justify your answer.
GC Rich DNA has more bonds AT Rich GC Rich Hydrogen bonds work like little magnets. The more bonds, the more attraction between the two strands
Melting temperature Proram the miniPCR for a linear ramp from 35 °-99°. This will cause the machine to slowly heat over time. Place tubes in wells. *Be careful. Lid and heat block will be hot.Every two minutes, transfer tubes to illuminator. Take picture and return to heat block.GOAL: Identify approximate melting temperatures for each tube
Programming the miniPCR Choose +NEW Choose Create Linear Ramp Protocol
Programming the miniPCR Give it an awesome and creative name Start @ 35 ˚ End @ 99˚ Time 10 min Save and run
Melting temperature Results?
pH and hydrogen bonds Alkaline (high pH) conditions have an excess of OH - ions in solution. The excess negative charges will be attracted to the positively charged hydrogen atoms in the hydrogen bonds. With the hydrogen atoms now bonding with the OH - ions in solution, the DNA will denature. OH OH OH OH OH
pH and hydrogen bonds Create a pH dilution series using NaOH to test the effect of alkaline conditions on hydrogen bonding between nucleotides.Is this effect reversible? How could you test that?
DNA concentration Fluorescent dyes are often used in the laboratory to measure DNA concentration. Because the dye only fluoresces when bound to double stranded DNA, an accurate concentration can be measured even in a complex mixture containing other molecules. In the laboratory, normally a tool called a fluorometer would give an accurate reading of fluorescence intensity.
DNA concentration Create a DNA dilution series to measure the concentration of the unknown DNA sample.
Thank You! Feedback? Questions? @miniPCR support@miniPCR.com