Bio 219 Week 1 Reagents

PHEW

What are the reagents used in chDNA isolation?

Resuspension buffer (ATL), Proteinase K, RNAse A, Lysis Buffer (AL), Ethanol, Wash buffer 1 (AW1), Wash buffer 2 (AW2), and DNA grade water

What is the important component of resuspension buffer (ATL)?

Sodium dodecyl sulfate (SDS)

What does SDS do?

Forms a cation bridge to bind DNA to the silica membrane in a spin column

Solubilize membrane proteins

Solubilize membrane phospholipids

Release DNA binding proteins

What is SDS?

Sodium dodecyl sulfate, a strong anionic detergent

How does SDS solubilize proteins?

By disrupting the lipid bilayer of gram negative cells and bringing proteins into solution as protein-lipid-detergent complexes

How does SDS help release the DNA binding proteins?

By:

Denaturing the DNA binding proteins and

Binding both membrane and non-membrane proteins and monomers

What is Proteinase K?

An enzyme that helps release nucleic acids and deactivate nucleases

What are nucleases?

Enzymes that degrade nucleic acids

What is the purpose of adding Proteinase K?

To degrade nucleases and prevent nucleic acids from nuclease attack

What is RNase A?

A ribonuclease that degrades RNA in a sample

How does RNase A work?

By:

Cleaving the 3’ of the phosphodiester bonds after pyrimidine (A or G) nucleotides in single stranded RNA

What effect does RNase A have on DNA?

DNA will remain intact since it doesn’t have the 2’ OH group that the RNase A needs to form the cyclic intermediate cleavage product

What key component does Lysis Buffer (Buffer ATL) have?

Chaotropic salt (guanidium chloride)

Name the chaotropic salt used in this experiment?

Guanidium chloride

What do chaotropic salts do?

Disrupt the regular H bond structures in water

Disrupt the hydrate shells of in vivo nucleic acids and the hydrophobic interactions between neighboring stacks of base pairs

Denature residual proteins

In vivo nucleic acids have what shell made of what?

A hydrate shell made of water molecules

What is the purpose of a hydrate shell?

To maintain the solubility of DNA in aqueous solutions

What is the result of using guanidium chloride on the sample in the experiment?

Conditions are set so the DNA can selectively bind to the silica resin in the transfer tube

What is the purpose of using ethanol in this experiment?

To enhance and influence the binding of nucleic acids to the silica resin

Aid lysis buffer in creating a more hydrophobic solution

What conditions does ethanol help create in the sample?

A more hydrophobic solution (more hydrophobic conditions)

What does Wash Buffer 1 (AW1) contain?

A low amount of chaotropic salts

What is the purpose of Wash Buffer 1 (AW1)?

To bind to and remove proteins and colored contaminants

What does Wash Buffer 2 (AW2) contain?

Ethanol

What is the purpose of Wash Buffer 2 (AW2)?

To remove the salts added from AW1

What does DNA grade water contain?

NO salts, DNases, or Proteases

What is the purpose of using DNA grade water?

To rehydrate and renature the DNA, which makes the DNA lose affinity for the silica

What is the order that the reagents are used?

Resuspension Buffer (Buffer ATL), Proteinase K, RNase A, Lysis Buffer (AL), Ethanol, Wash Buffer 1 (AW1), Wash Buffer 2 (AW2), and DNA grade water

SDS dissociates in the presence of?

Chaotropic salts (ex. from the lysis buffer)