genomes Flashcards
what is pcr
polymerase chain reaction
explain the process of pcr
used to select a fragment of dna(containing the gene or bit of DNA you’re interested in) and amplify it to produce millions of copies in just a few hours.
stage 1 of pcr
reaction mixture is set up that contains he DNA sample, free nucleotides, primers and DNA polymerase
what are primers
short prices of DNA that are complementary to the bases at the start of the fragment you want
stage 2 of PCR
DNA mixture is heated to 95 degrees to break the hydrogen bonds between the two strands of DNA. polymerase doesn’t denature even at this high temperature
why is it important that DNA polymerase doesn’t denature when heated to 95 degrees
means many cycles of pcr can be carried out without having to use new enzymes each time
stage 3 of PCR
mixture is now cooled to between 50-65 degrees so that the primers can bind (anneal)to the strands
stage 4 of PCR
reaction mixture is heated to 72 degrees so DDNA polymerase can work
stage 5 of PCR
DNA polymerase lines up free DNA nucleotides alongside each template strand. complementary base pairing means new complementary strands are formed
stage 6 of PCR
2 new copies of the fragment of DNA are formed and one cycle of PCR is complete
stage7 of PCR
the cycle starts again, with the mixture being heated to 95 degrees and this time all four strands(2 original and two new) are used as templates
number of fragments in each pcr cycle
1st cycle=2x2= 4 DNA fragments
2nd cycle=4x2=8 DNA fragments
3rd cycle=8x2=16 DNA fragments
what is electrophoresis
uses an electrical current to separate out DNA fragments, RNA fragments, or proteins depending on their size
first step of electrophoresis
add a gel tray to a gel box(agarose gel)
leaves to solidify
row of wells created, closest to negative electrode
add buffer solution to the reservoirs at the sides of gel box, surface of he gel becomes cover in buffer solution
second step of electrophoresis
use micro pipe tee to add same vol loading dye to each well (helps visibility)
add a set volume of a dna sample to first well repeat process in other wells
