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9

This diagram would be easier for you to conceptualize if you instead used the actual nucleotides in the restriction sites and green GGAGAA region along with your gene of interest (GOI). I recommend working with the DNA sequences when learning about primer design for various applications. I believe it has led you to be slightly confused with what is the ...


5

This paper describes an allele-specific PCR protocol to detect CAG repeat expansion in MJD patients: Maciel P, Costa MDC, Ferro A, et al. Improvement in the Molecular Diagnosis of Machado-Joseph Disease. Arch Neurol. 2001;58(11):1821–1827.


5

How does DNA synthesis from the forward primer stop when it reaches the point where the reverse primer is? Briefly, it doesn't. You get a lot of single-stranded long pieces of DNA (step 1 here, notice the overhang ends). But the exponential part of PCR only amplified common overlapping regions in these long single strands, also known as your target region ...


4

They don't stop replication. It's just that you replicate your dna in a number of cycles, let's say 30. after which you'll have a approximatly 10^6 molecules of the amplified region and only a few longer strands. If you draw it out you'll see that your region of interest quickly start to dominate. original strand ----/ /----ppp-------------------------/ /--...


4

In order to assemble your TAG oligos by PCR you will need to redesign your primers so they are complementary to the TAG oligos as DNA polymerases work by adding nucleotides to the 3' end of a DNA strand. Your design would look like this : Now with this design you won't be able to assemble everything together as TAG-3 complementarity to primer 2 only ...


4

I TRY to answer. First of all, no we cannot be 100% sure that the primers are effective against all strains. However, the strains that are under media attention right now are due to variants in the spike protein, while RT-PCR primers are designed on different genes. One WHO document (I do not know if it's the official guideline) targets RdRp (RNA-dependent ...


3

Well, technically yes but no. Why yes is because on paper this looks simple enough but think about the technicalities. Your FP1 and FP2 will have different TMs, meaning the temp required to make the complete fragment with FP1 and RP1 will be different than to the MP of FP2. Effectively when the temp for FP2 reaches it might cause FP1 and RP1 to detach.Or the ...


3

You could add the overhangs with PCR, then gel purify or PCR purify. I don't think it will pose a big problem as I've done it before and all of the colonies I screened were correct.


3

I suspect mispriming due to annealing temp being too low compared to predicted melting temp of overlap regions. As the protocol in your link notes: (3) Choose annealing temperature wisely. We recommend to use the same as min_Tm by Primerize design, which is usually between 60-64 °C. (4) Check PCR product on 4% agarose gel. If assembly is unsuccessful with ...


3

This is a bit complicated from far away, but I will share a few thoughts on it. The occurence of primer dimers depends on the design of the primers (sometimes they are inevitable due to sequence limitations), but then they also show up in the negative controls. If they are not present in the negative controls (meaning: never), I would not expect them to be ...


3

Gibson Assembly is not ideal for short fragments; chances are that the T5 Exonuclease will digest your entire fragment before it has the chance to hybridize with the backbone. For fragments shorter than 200 bp NEB recommends a 5-fold excess to compensate for this, but in your case the fragment would only be around 130 bp long. It might work, but efficiency ...


3

No, some strains contain mutations causing partial or complete failure of amplification of at least one of the test targets. Here's an FDA bulletin on the issue from January: https://www.fda.gov/medical-devices/letters-health-care-providers/genetic-variants-sars-cov-2-may-lead-false-negative-results-molecular-tests-detection-sars-cov-2 As the bulletin notes, ...


3

Summary The systematic chemical names of many important biological molecules are too long to write routinely in full, and in any case were preceded historically by abbreviated forms which generally stressed the distinguishing features of importance to those studying their metabolism. Thus, as the poster points out, a molecule such as dATP (image below) has a ...


3

There might be a confusion of terminology. There is real-time quantitative PCR, which can sometimes be called RT-PCR. There is also reverse-transcriptase PCR, which is also confusingly called RT-PCR. To try to reduce confusion, real-time quantitative PCR is also called RT-qPCR or qPCR. What you describe in your question is the reverse-transcriptase type of ...


2

I just wanted to elaborate on the previous answer regarding the confusion between real-time (quantitative) and reverse-transcription(RT) PCR. RT-qPCR refers to real-time quantitative PCR, but some organizations refrain from any reference to quantitation (because it's really only semi-quantitative), instead using the nomenclature rRT-PCR (reverse ...


2

This is done because at some point you start amplifying nonsense and not get a meaningful signal. If (and this is most often not the case) you have the ideal doubling of you DNA template in each cycle, you can calculate how little DNA you start with to detect it only around cycle 40 - or the other way round: What you will amplify and see as a "signal&...


2

In an open system, "mRNAs are detected because of their property of being expressed in the tissue sample isolated for study. This approach is in contrast to what are called closed-system approaches, in which the mRNAs to be detected are selected as candidates in advance of analysis; a contemporary example of a closed system would be a gene “chip” ...


2

I like your hand-drawn figure. I will do a pictorial explanation starting from that. You are correct in the primer binding and first stage of PCR step. But PCR1 and PCR2 and the final product is wrong. This is the way you think about it - In each step the primers bind to products extended in the previous steps (+ original template DNA, which is miniscule in ...


2

I made the large-scale in a falcon tube and then moved the mixture to PCR tubes each containing 50 μL, so the PCR conditions should be the same as the single PCR amplification If all reagent concentrations and cycling conditions are the same, I suspect that you did not sufficiently mix your large-volume reaction prior to dispensing as 50 μL aliquots. It ...


2

PCR is highly specific (meaning it amplifies only the DNA the primers bind to), so the results in general are good. But: Due to the exponential nature of amplification, even very small mispairings can lead to the amplification of a very small amount of DNA and subsequently to a fluorescent signal. There are basically two ways of getting this amplification (...


2

I basically agree with Bob, but wanted to add a few more details. Out of the options that you have available, option 4 is the most common way to do it. I wouldn't consider options 1 and 3. I don't think option 1 will really work to produce the amount of plasmid that you need, option 2 is a much easier way of amplifying it. As for option 3, you shouldn't need ...


2

Option 2 is your simplest and easiest. It should give you unlimited plasmid to work from. Note that ordering pre-made sequences of this size isn't particularly cheap and might take a few weeks to get to your destination. It may be easier (quicker + cheaper) to find a plasmid with the luciferase CDS and terminator already built in. You could then amplify this ...


1

While @Chris's answer is correct, there is a complication that comes into it: The principle behind quantitative PCR (qPCR) is that you have a reaction which, if your reaction is 100% efficient, doubles the number of DNA strands produced at each cycle. In theory, you can start from one strand and get 2, then 4, then 8 in a 2n style equation, where n is the ...


1

Actually, neither A nor B in the example are a correct interpretation of a false positive rate, because there isn't enough information provided to determine a false positive rate. In the terminology of clinical diagnostic testing evaluation, testing outcomes can be classified into one of four fundamental categories: True positive, false positive, true ...


1

Yes, this is perfectly fine. Many companies sell PCR Master Mixes (granted, without primers): Thermo Fisher Sigma-Aldrich BioRad Promega Qiagen If you choose to make your own, make sure it's stored at -20°C, and be sure to aliquot it so it's not going to be subjected to multiple freeze/thaw cycles. Obviously you should mix it well before aliquoting, but ...


1

It is used to determine whether the unused/excess amount of plasmid has contaminated the final solution of the desired recombinant DNA or not. Electrophoresis is performed on the "Blank"(uncut) plasmid to define its bands.If the electrophoresis result contains the bands of both "blank" and recombinant DNA , it shows that your solution ...


1

Real-time PCR is used to quantify a PCR target by measuring some indicator of target amplification in real time after each amplification cycle, thus it is sometimes(often) called quantitative PCR (qPCR). The key to distinguishing between reverse transcription PCR and real-time PCR in the context of a paper or protocol is to look at the starting material ...


1

are the primers used in every cycle the same sequence? Yes. You put the whole reaction on the thermocycler, and then walk away until all the cycles are complete. But one could do a second round of PCR with different internal primers on that first PCR product, if you wanted. Second, why are primers not reusable for different cycles? Are you asking why you ...


1

The primer is incorporated into the start of the new strand. The number of dna strands produced is limited by the number of primers.


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