PCR

Question 1

what is PCR

Answer 1

Polymerase chain reaction amplifies fargments of DNA by in vitro enzymatic replication

Question 2

what does pcr amplifie

Answer 2

• single gene
• specific section of gene
• non-coding section of ADN

Question 3

What u need

Answer 3

DNA template (sample)
Primers
DNA polymerase at 70 grades (thats why we need heat)
dNTPs (needed for the polymerase)
Buffer solution (right environment)
dicalent cations
monovalent cation

Question 4

Reaction tubes

Answer 4

allow heat to penetrate effectively with thin walls (thermal cycle)
15-100 ul
for enzymes to work

Question 5

first step (denaturation)

Answer 5

hold 95 degress for five minutes
mixture with primers nucleotides dna starnd and dna polym,erase is heated to 93 degrees few seconds
this breaks the dna strande in two single bands

Question 6

second step (annealing)

Answer 6

the two strands are cooled down to 55 degrees

which allow de primers to attach to the 3’ of the sequences

Question 7

third step (elongation)

Answer 7

the mixture is heated to 72 degrees
this is the temperature that allows the polymerase to attach to the primers and to start replicating dna with the nucleotides already added previously
keep going to finalise production of dna
-final hold 4-115 degress for short term storage

Question 8

what happens after each cycle

Answer 8

products doubles (exponential amplification)

Question 9

how many times is the process repetead

Answer 9

around 35

Question 10

gel electrophoresis

Answer 10

gel separation

agarose gel to find what organisms are present in the sample

Question 11

gel electrophoresis technique

Answer 11

sample are inserted in well in the gel in the tank of the machine where a current of electricity is transmitted which allows the DNa to move through the gel
once separated, ultraviolet light is used to visualise the DNa

Question 12

how are samples obtaines

Answer 12

obtain dna and mix into sample
incubate at 56 for 10 min, bigurous agitation
incubate at 100 for 6 min, vigorous agitate
centrifugate for 5 min

Question 13

Dna strands united by

Answer 13

hydrogen bonds (broken in denaturation)