Flashcards in Biotechnology (W16) - Week 3-4 Background Deck (38)
To perform analysis by southern blotting, DNA is initially what?
Digested by one or more restriction endonucleases.
Restriction digest can be performed on which DNA types?
In this lab, we will be performing a restriction digest of what type of DNA?
Digested gDNA is then separated by what method?
Agarose gel electrophoresis
The nucleic acids contained in the gel, post restriction digest, are ______ then transferred to what?
The nucleic acids contained within the gel are denatured and transferred to a positively charged nylon or nitrocellulose membrane.
What transfer method will we be employing this lab?
Capillary transfer approach
In terms of reliability, efficiency and speed, how would one describe transfer by capillary action?
Most reliable and efficient in terms of transfer.
Not the best in terms of speed.
How does the capillary transfer approach work?
Blotting papers are used to wick transfer buffer through the gel, through the membrane, and into a stack of fibrous paper towel.
Eventually, either the wick becomes saturated with transfer buffer or the reservoir becomes exhausted.
As this occurs, the gel becomes dehydrated and the DNA is drawn out.
The nucleic acids are carried from the gel to the membrane where, movement is arrested due to attraction between the positively charged membrane and negatively charged DNA.
The rate of transfer of the DNA fragments (using capillary transfer) depends on what?
The molecular weight of the fragment and concentration of agarose within the gel.
Smaller DNA fragments are transferred from the 0.8% agarose gel to the membrane within ______ whilst larger pieces are transferred ________ (more than one word).
1. 1 hour
2. more slowly and less efficiently
What is the concentration of the gel?
How is the efficiency of transfer of larger DNA fragments accomplished?
Acid hydrolysis prior to denaturation. Essentially, larger pieces are hydrolyzed into smaller pieces, resulting in greater ease and speed of transfer.
The resulting membrane contains a replica of the _________ originating from the _________ ___.
2. agarose gel
The positively charged membrane allows for what with the nucleic acids?
Electrostatic interactions with the negatively charged nucleic acids.
What must also be done to the transferred DNA to ensure that the ssDNA fragments are completely immobilized on the membrane?
Once the DNA is permanently bound, what is done to remove background interactions for chemiluminescence?
Pre-hybridization with a non-specific DNA and protein such that all unoccupied sites are covered/blocked from non-specific binding by probe molcules.
To pre-hybridize, what commercially prepared product will we be using?
The final step of the Southern blot is what?
the hybridization of a labeled probe to the digested DNA at specific salt, pH, temperature and nucleic acid concentrations.
What is the non-specific DNA in the rapid Hyb product? (milked em myself)
salmon sperm (Fkn nice m8)
What is the non-specific protein in the rapid Hyb product?
Typically, formamide is used in the hybridization buffer for what reason?
To destabilize the secondary structures of ss nucleic acids.
Formamide effectively does what to the DNA and probe?
Lowers the effective melting temperature of the DNA and probe and permits lower hybridization temperatures from 65-68C.
What is the purpose of polyethylene glycol?
Used to increase the speed of probe hybridization by reducing the concentration of the water in the hybridization solution.
What does polyethylene glycol effectively do (by reducing the water content in the hybridization solution)?
increases the concentration of the biotin labeled probe and promotes faster hybridization
What factors determine the DNA melting temperature and directly affect the binding of ssDNA probe to a complementary sequence immobilized on the membrane?
Temperature and ionic strength of the hybridization solution.
What is the stringency of the reaction?
During nucleic acid hybridization or reassociation, the strictness to which base pairing is required under specific conditions of temperature, pH, salt concentration, etc.
Conditions of high stringency would be what?
Requires all bases of one polynucleotide to be paired with complementary bases on the other.
Conditions of low stringency allow what?
Some bases to be unpaired (between probe and DNA)
What are permissive conditions of DNA hybridization?
low temperature and high salt concentrations.