miniprep & pcr

Question 1

purpose of miniprep

Answer 1

amplify and isolate purified plasmid

Question 2

step 1

Answer 2

OVERNIGHTS - allows e.coli to replicate (amount of plasmid & insert increases), LB - ampicillin rich medium to grow e.coli

Question 3

labelling clone

Answer 3

School # (19) initials clone # . year

Question 4

step 2

Answer 4

CENTRIFUGE - bacteria cells will collect in a pellet, the supernatant is the LB Ampicilin

Question 5

step 3

Answer 5

RESUSPEND PELLET & P1 BUFFER - do not want any enzymes

Question 6

step 4

Answer 6

ADD BUFFER P2 - sample becomes clearer from denaturing proteins, centrifuge again

Question 7

step 5

Answer 7

ADD BUFFER N3 - neutralize pH

Question 8

step 6

Answer 8

CENTRIFUGE CELL DEBRIS - white pellet will form (precipitate)

Question 9

step 7

Answer 9

TRANSFER SUP TO SPIN COLUMN - pour supernatant into spin column

Question 10

step 8

Answer 10

CENTRIFUGE SPIN COLUMN

Question 11

step 9

Answer 11

WASH COLUMN WITH BUFFER PE - remove nucleases, maintains pH, affects getting sequence if not cleansed thouroughly

Question 12

step 10

Answer 12

REMOVE ALL BUFFER PE - spin column 2nd time

Question 13

step 11

Answer 13

ELUTE DNA WITH BUFFER EB

Question 14

what buffers are used during minprep

Answer 14

5; P1, P2, N3, PE, EB

Question 15

what does pcr stand for

Answer 15

polymerase chain reaction

Question 16

principle behind PCR

Answer 16

fragment of DNA repeatedly synthesized using DNA polymerase, particular sequence is AMPLIFIED billion fold or more

Question 17

components of PCR

Answer 17

taq polymerase, template DNA, dNTPs, oligonucleotide primers, thermal cycler

Question 18

taq polymerase significance

Answer 18

heat stable enzyme, resist denaturation

Question 19

template DNA function

Answer 19

sample that contains region to amplify

Question 20

dNTPs function

Answer 20

monomers taq polymerase uses to synthesize new DNA

Question 21

PCR: Initial Denaturation

Answer 21

place in microfuge tube at heat for 5 min at 95 C –> disrupts H Bonds, denatures double stranded to single stranded DNA

Question 22

how many time do you amplify DNA in PCR

Answer 22

30x

Question 23

PCR: Amplification

Answer 23

denature target DNA and break H Bonds (single stranded DNA - 1 min 94 C) (primer annealing - 1 min 50 C) (synthesis - 1 min 72 C)

Question 24

PCR: Additional Elongation

Answer 24

maes sure DNA strands are synthesized in full length, 5 min 72 C, 2-3 hours = billion copies of template DNA

Question 25

what is the longest stage of ocr, why

Answer 25

amplification, 30x for 5 min each (2 - 3 hours)

Question 26

purpose of CaCl

Answer 26

weakens bacteria membrane