practical - TB

Question 1

what does PCR stand for *

Answer 1

polymerase chain reaction

Question 2

what is the purpose of PCR *

Answer 2

amplify DNA so we can see it

amplify specific types of DNA

Question 3

how does PCR work *

Answer 3

heat up DNA so it denatures - the double strand falls apart - 1 minute at 94degrees

cool down with forward and reverse primers that anneal on the broken DNA - 45 seconds at 54degrees

raise the temperature

the primer extends and makes more DNA chains - 2 minutes at 72 degrees - only have dNTPs

Question 4

what are the components needed for PCR *

Answer 4

nucleotides

dna

forward adn reverse primers

DNA polymerase

buffer - make ph and salt so PCR can work

Question 5

outline of how PCR is performed *

Answer 5

set up a PCR reaction - 10mins

run PCR in thermocycler - 2hrs

prepare agarose gel for analysis of PCR producrs - 10 mins and 30mins to solidify

agarose gel electrophoresis to separate the PCR product 30-45mins

visualise and image PCR products - 10mins

Question 6

what is in agarose gel *

Answer 6

agarose powder

water

SYBR safe DNA stain - you excite this with the blue light so that you can see the DNA

buffer - Tris Acetic acid EDTA (TAE)

Question 7

how many g of agarose re in 40ml of a 1% agarose mixture

Answer 7

0.4g

Question 8

how many g of agarose are in 40ml of a 2% conc of agarose mixture

Answer 8

.8g

Question 9

how much SYBR safe is added to 40ml agarose at 1% conc

Answer 9

the dilution of SYBR safe was 1:20000

therefore

40/20000 = 2ul

Question 10

why is the agarose slightly pink

Answer 10

because of the SYBR safe dye

Question 11

why do you add 12.5 5x concentrated DNA loading buffer containing a 50ul PCR reaction *

Answer 11

because that would make the total vol 62.5, therefore 1/5 of the vol is the loading buffer - it is necessary for it to be 1/5 of vol because it is concentrated 5x

Question 12

what difference will it make to use a 1% or 2% agarose gel and which will be better for this application *

Answer 12

higher percentage is better to separate the small pieces of DNA

however higher percentage means that the gel runs really slow

therefore 1% not ideal because worse separation - but faster that is why it is ised

Question 13

what is the purpose of adding DNA loading dye to sample *

Answer 13

adds glycerol so makes the sample heavy so that the DNA moves to the bottom of the well

there is blue dye in the loading dye so you can see where the DNA has travelled

Question 14

which direction does dna move and why *

Answer 14

move from -ve to +ve because phosphate backbone is negative

Question 15

do smaller or bigger DNA fragments move faster *

Answer 15

smaller

Question 16

how will you know if the DNA fragment is the correct size *

Answer 16

using the DNA ladder

Question 17

what do you expect to see on the gel if 1 pt is infected with TB and the other isnt *

Answer 17

positive control have band at 30bp

so will pt 1

pt 2 will not

Question 18

what do you expect to see if pt 1 is infected with TB and 2 is infected with M bovis *

Answer 18

bands on both - the dna sequences cannot be differentiated

Question 19

what are some of the limitations of PCR *

Answer 19

that a prior knowledge of the DNA sequence is required

that not all DNA sequences can be differnetiated so it might not be able to differentiate between different diseases

that some mutations might be in conserved areas so not able to be identified using PCR

Question 20

how can you make PCR more specific *

Answer 20

need to change the primers so that they only anneal to DNA that is specific to what you are looking for

Question 21

what are the advantages of using PCR to culture based methods *

Answer 21

faster

safer - dontbhave to amplify the whole organism

Question 22

can PCR be used to determine resistance *

Answer 22

yes sometimes if caused by specific genes, not if they are in the highly conserved parts of the genes

Question 23

is PCR used in diagnostic labs *

Answer 23

yes eg for clamydia and viral infection

Question 24

what are other applications of PCR *

Answer 24

food quality control

criminology

DNA testing

to detect infection