Flashcards in Really crappy biotech cue cards for last minute studying before midterm Deck (23)
The resulting membrane, after capillary transfer contains what?
A replica of fragments originating from the agarose gel.
What is the additional insurance used to immobilize DNA on the membrane?
Once the DNA is permanently bound, what is added to block it?
Salmon sperm DNA as the non-specific DNA source and skim milks as the inert protein.
What is the point of blocking?
Inert proteins will occupy unbound sites to prevent background interactions.
What are we using to block the membrane?
What is the final step in the Southern blot?
Hybridization of a labeled probe to the digested DNA at a specific salt, pH, temperature and nucleic acid concentration.
What is the purpose of adding formamide?
Used in the hybridization buffer to destabilize the secondary structures of ssDNA.
What does the destabiliation afforded by formamide due?
Lowers the effective melting temperature of DNA and the probe, and permits lower hybridization temperatures from 65-68C.
What is the purpose of polyethylene glycol?
Increase the speed of hybridization by reducing the concentration of available water in the hybridization solution.
What does polyethelyne reducing water content effectively do?
increases the concentration of the biotin labeled prone and promotes faster hybridization.
What determines DNA melting temperature and directly affects the binding of the ssDNA probe to complementary sequence immobilized on the membrane?
Temperature and ionic strength of the hybridization solution.
The conditions of temperature and ionic strength in the hybridization solution are refferred to as?
the stringency of the reaction.
What is high stringency?
high temperature and low ionic strength which results in a need for perfect complimentarity for binding.
What is low stringency?
Low temperature and high salt concentration which results in not needed perfect DNA alignment.
The detection of the biotin labeled probe is accomplished through?
visualization of the interaction with streptavidin via a chemiluminescent detection system.
The streptavidin chemiluminescence system is very sensitive allowing for?
detection of picomoles of hybridized probe.
Following hybridization, the blot is exposed to what?
streptavidin bound to HRP.
Streptavidin binds to biotin causing permanent interaction with the ___DNA probe.
Visualization occurs because?
An enhanced luminol and oxidizing agent are added. Luminol interacts with HRP when oxidized and that results in light.
Light solely comes from?
the probe binding to the target sequence.
When the luminescent image is captured, what is interpreted?
MW and number of bands.
The results will be used to identify if the digested DNA contained what?
The sequence of interest.