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Nested PCR Assay: PCR was per formed on the DNA extracted from all the samples (n-238): tWO pairs of species-specific primers (Table I) were used t0 amplifv &a...

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Nested PCR Assay: PCR was per formed on the DNA extracted from all the samples (n-238): tWO pairs of species-specific primers (Table I) were used t0 amplifv & region (438 bp) of the 27 kDa out- er membrane pfotein (coml) gene (12). Each 25 pL reaction consisted of 12.5 AL master mix . AM of

Nested PCR Assay: PCR was per formed on the DNA extracted from all the samples (n-238): tWO pairs of species-specific primers (Table I) were used t0 amplifv & region (438 bp) of the 27 kDa out- er membrane pfotein (coml) gene (12). Each 25 pL reaction consisted of 12.5 AL master mix . AM of



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A. If the PCR shown in Figure $10-12$ is carried through an additional two rounds of amplification, how many of the DNA fragments (gray, green, red, or outlined in yellow) will be produced? If many additional cycles are carried out, which fragments will predominate? B. Assume you start with one double-stranded DNA molecule and amplify a 500 -nucleotide-pair sequence contained within it. Approximately how many cycles of PCR amplification will you need to produce 100 ng of this DNA? 100 ng is an amount that can be easily detected after staining with a fluorescent dye. (Hint: for this calculation, you need to know that each nucleotide has an average molecular mass of $330 \mathrm{g} / \mathrm{mole} .$

In here as if the PCR shown in this figure here is carried through an additional two rounds of applicant fate. Amplification rather how many of the DNA fragments labeled in great green or red or outlined in yellow are produced, so there will be a total of two gray to green for red on 22 yellows. This is because the DNA fragment in yellow increases exponentially and will eventually overrun the reaction phrases products rather.

Okay, so for this particular problem, just to go over what they're asking of us, you're trying Teoh PCR a piece of DNA. And we've got two primers, this one on the top that mark appear with a green dot and this one in the bottom that I murdered down here with a blue dot um, And when we run PCR, we're expecting, you know, to see these nice bands separated from each other. Instead, we see a smear that's about 25 to 30 base pairs. So why is that? So when you look at these to primers together, there's a reason I set this up this way, and it's because the primers actually overlap at their ends. So these lines that I'm drawing or not just for fun, each one of these has a complimentary base pair on the other side that they can bond with. So what's probably happening is that the primers are a kneeling to one another instead of the sequence that you're trying to get them Teoh to Aneel with. So by doing this, they're not binding to the DNA sequence that you're trying to get them divine to their Vinings one another and if you look even closer, this from this to the end of this, that's exactly 25 base pairs. So by counting issue number one of these which you can dio, um you will see that it pretty much matches with the results that you're getting this this 25 30 base pair smear on your electrophoresis shall. Okay, so issue here again is that the primers have overlap where they can and Neil to one another because of the complementary base pairs.

Counsel for the question here. Primer. We have to design to a primer here. Primer one five c c t c g A g d. See a BC G 80 d C t g three and we HAVE Raimond to fly five c g c g C A C A D C e g A gsc g A C C A three

And so for the question here, right, These sequences of the two 12 residues primary state could be used to amplify the loving DNA segments by PCR. So here, the Cuban segment we have here first look at the first of all types and the complementary basis of the last. Well, nobody's do know these segments of the primaries to be used. So here we have Eddie Mm G g c A D A G see and C d g a c c e g c g g c g c c. So look at the 1st 12 nucleotides and the complementary basis of the last 12 nucleotides to know the sequences of the primaries to be used.


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