Exam 3 Chapter 10 Flashcards
(33 cards)
PCR initial denaturation
94-95* for 10-15 minutes
PCR cycles temperature
~55*
PCR final extension
~72* for 10 minutes
PCR hold
~4*
acts as a fridge for DNA
PCR cycles - denaturation
95* for 1 min
PCR cycles - annealing
attach primers forward and reverse
temp will vary - 55-65*
PCR cycles - extension
72*
number of cycles in PCR
20-50
over 50 cycles will use too much reagent
products of each cycle are used to
serve as the DNA template for the next cycle
each cycle ____ the amount of DNA synthesized in the previous cycle
doubles
RT-PCR
reverse transcription polymerase chain reaction
involves isolation of mRNA from a cell, using reverse transcriptase to convert it to cDNA, then amplification (cloning) of the DNA via PCR
then gel electrophoreses
uses of RT-PCR
detect and quantify RNA
study gene expression - compare it in different cells
1-step RT-PCR
all reagents go in one tube
reverse transcriptase added (enzyme)
add buffers, water, primers, polymerase, DNTPS
RT-PCR protocol
reverse transcription using RT enzyme
initial PCR activation
cycles
final extension
hold
RT-PCR - reverse transcriptase using RT enzyme
50* C for 30 min
RT-PCR - initial PCR activation
95* for 15 min
number of cycles for RT-PCR
35-50
RT-PCR - final extension
72* for 10 min is most common, but can involve trial and error
RT-PCR - hold temp
4*
limitation of RT-PCR
qualitative, not quantitative
don’t know by what factor genes are upregulated
SCVs
small colony variants
produced by some, not all, bacteria
auxotroph
mutant form
ex. lipoic acid -> cofactor for acetyl CoA to enter
lip A mutant -> no acetyl CoA
smiley effect
comes from the ladder of molecular markers sliding over to the side - forming a “smiley”
sometimes skip MM as a result
qRT-PCR
allow level of gene upregulation to be quantified
used in published research for more precision
uses a fluorescent probe
