practical - TB Flashcards
what does PCR stand for *
polymerase chain reaction
what is the purpose of PCR *
amplify DNA so we can see it
amplify specific types of DNA
how does PCR work *
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
what are the components needed for PCR *
nucleotides
dna
forward adn reverse primers
DNA polymerase
buffer - make ph and salt so PCR can work
outline of how PCR is performed *
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
what is in agarose gel *
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)
how many g of agarose re in 40ml of a 1% agarose mixture
0.4g
how many g of agarose are in 40ml of a 2% conc of agarose mixture
.8g
how much SYBR safe is added to 40ml agarose at 1% conc
the dilution of SYBR safe was 1:20000
therefore
40/20000 = 2ul
why is the agarose slightly pink
because of the SYBR safe dye
why do you add 12.5 5x concentrated DNA loading buffer containing a 50ul PCR reaction *
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
what difference will it make to use a 1% or 2% agarose gel and which will be better for this application *
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
what is the purpose of adding DNA loading dye to sample *
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
which direction does dna move and why *
move from -ve to +ve because phosphate backbone is negative
do smaller or bigger DNA fragments move faster *
smaller
how will you know if the DNA fragment is the correct size *
using the DNA ladder
what do you expect to see on the gel if 1 pt is infected with TB and the other isnt *
positive control have band at 30bp
so will pt 1
pt 2 will not
what do you expect to see if pt 1 is infected with TB and 2 is infected with M bovis *
bands on both - the dna sequences cannot be differentiated
what are some of the limitations of PCR *
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
how can you make PCR more specific *
need to change the primers so that they only anneal to DNA that is specific to what you are looking for
what are the advantages of using PCR to culture based methods *
faster
safer - dontbhave to amplify the whole organism
can PCR be used to determine resistance *
yes sometimes if caused by specific genes, not if they are in the highly conserved parts of the genes
is PCR used in diagnostic labs *
yes eg for clamydia and viral infection
what are other applications of PCR *
food quality control
criminology
DNA testing
to detect infection
