Flashcards in Library screening Deck (8)
What is the first step in screening a DNA library?
Identification of the E. coli plaques that contain the DNA sequence of interest
What are the 2 different ways the DNA sequence can be identified?
The pure sequence itself
The gene product- proteins or enzyme activity
Explain how an amylase gene could be screened for
Plate the library on a starch-containing agar plate
Incubate overnight and flood the plate with I2 and KI \
The clone containing amylase will have a clear ZOI where starch digestion has occurred
Describe antibody screening
Purify the protein of interest
Raise antibodies to that protein by injecting into rabbit
Plate the library on agar plate
Transfer library to filter
Lyse cells to release the proteins
Add the inert protein (usually bovine serum albumin) to block any non-specific binding
Incubate with radioactive antibody
Expose to X-ray film and radioactive antibody will blacken the film
Describe DNA hybridisation screening
Plate library on agar
Transfer to filter
Lyse cells and denature DNA with alkali
Add salmon sperm DNA to block non-specific binding sites
Incubate with radioactive probe
Expose to X-ray film
What is DNA hybridisation screening used for?
cDNA or genomic libraries
Where can DNA hybridisation screening take place?
With target DNA attached to nylon or nitrocellulose filter