BLD 430 EXAM 2 QUESTIONS AND ANSWERS WITH
COMPLETE SOLUTIONS VERIFIED GRADED A++
use of southern blot
DNA analysis
use of northern blot
RNA analysis
use of western blot
protein analysis
hybridization
binding with complementary structure
DNA probes
specific sequence synthesized in lab
RNA probes
transcribed from a synthetic DNA
protein probes
antibody
probe label
allows for visualization of hybridization
examples:
-radioactive 32P on probe
stringency
, strictness for the specific complementary sequence
high stringency
this means the probe binds to the specific target more correctly. this requires the
probe/target complementarity and longer probe. if the hybridization setting has TOO
HIGH stringency, the probe will not bind to the target.
low stringency
this does not require a long probe nor strong complementarity. if the hybridization
setting has too low stringency, the probe binds to non-target
factors affecting stringency
-temperature: high heat can provide denaturation of non-specific binding
-salt concentration of hybridization buffer: high salt concentration forces nucleic acid
strand closer together that can be helpful for hybridization
-denaturation concentration in buffer: denaturant separate DS DNA that allows probe to
bind
-length and characteristics of probe: long probe and/or having more G-C% makes probe
to bind easily even in high stringency condition
melting temperature (Tm)
optimal temperature to separate double stranded nucleic acid for hybridization. the
temperature where number of DS nucleic acid and single stranded nucleic acid are
same is set as tm
Tm calculation based on # of GC pair and # of AT pair for short sequence
Tm= 4C x number of GC pair + 2C x number of AT pair
sequence complexity (C0t)
COMPLETE SOLUTIONS VERIFIED GRADED A++
use of southern blot
DNA analysis
use of northern blot
RNA analysis
use of western blot
protein analysis
hybridization
binding with complementary structure
DNA probes
specific sequence synthesized in lab
RNA probes
transcribed from a synthetic DNA
protein probes
antibody
probe label
allows for visualization of hybridization
examples:
-radioactive 32P on probe
stringency
, strictness for the specific complementary sequence
high stringency
this means the probe binds to the specific target more correctly. this requires the
probe/target complementarity and longer probe. if the hybridization setting has TOO
HIGH stringency, the probe will not bind to the target.
low stringency
this does not require a long probe nor strong complementarity. if the hybridization
setting has too low stringency, the probe binds to non-target
factors affecting stringency
-temperature: high heat can provide denaturation of non-specific binding
-salt concentration of hybridization buffer: high salt concentration forces nucleic acid
strand closer together that can be helpful for hybridization
-denaturation concentration in buffer: denaturant separate DS DNA that allows probe to
bind
-length and characteristics of probe: long probe and/or having more G-C% makes probe
to bind easily even in high stringency condition
melting temperature (Tm)
optimal temperature to separate double stranded nucleic acid for hybridization. the
temperature where number of DS nucleic acid and single stranded nucleic acid are
same is set as tm
Tm calculation based on # of GC pair and # of AT pair for short sequence
Tm= 4C x number of GC pair + 2C x number of AT pair
sequence complexity (C0t)
