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12

Too long for a comment: I would say it depends on the PCR. I have personally used maximum volumes of 50ul, if I needed to prepare higher volumes, I made one mix which subsequently got distributed to more tubes with 50ul max. The problem with big volumes is to get a fast and homogeneous heating and cooling. If the outside of the tube is faster, the reaction ...


12

(Also too long for a comment.) This question reminded me about an anecdote told by Arthur Kornberg concerning the approach to scaling up that was adopted by Reiji Okazaki (discoverer of Okazaki fragments). Here is a quotation taken from For the Love of Enzymes: The Odyssey of a Biochemist by Arthur Kornberg   Reiji’s research style is not readily ...


11

Here's some step-by-step advice on good pipetting technique: Make sure the pipette is set for the correct volume Ensure the tip is firmly attached Keep the pipette vertical when pipetting Slowly and smoothly depress the plunger to the correct point (the first stop position) Insert the tip in to the liquid to be pipetted. Ensure the tip is properly immersed ...


10

I would draw the line beyond 35, but thats a bit cosmetic. The reasons are manyfold: due to the exponential fashion of the amplification (ideally) reagents are used up at some point reagents degrade, this is especially true for the dNTPs the activity of the enzyme, despite being heat-stable is declining over time beyond 35 cycles the exponential curve is ...


9

Instead of searching for the stability of dNTPs, consider the stability of their constituent parts. Caveat: ignoring the bonds between the different parts of dNTPs likely misrepresents the stability of the intact molecule, given that the covalent bonds between the nucleobase, sugar, and phosphate pieces each have their own relative stabilities. Hydrolysis ...


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 ...


7

I don't know the exact half-life of this special Pfu polymerase, but generally Pfu polymerases are pretty stable. This source gives a half life of 18-25 hours at 95°C, meaning you keep the enzyme at 95°C for the whole time a you still have 50% activity after that time. Since you don't do that, I wouldn't worry too much about the activity. To be safe, you can ...


7

How many cycles of PCR before dNTPs run out? Assume a 25 μl reaction. Assume 200 μM dNTPs. 200 μM dNTPs = 200 pmol μl -1 so in 25 μl reaction, there are 5000 pmol of dNTPs = 5000 x 10-12 x 6 x 1023 molecules = 3 x 1015 molecules dNTP Assume that we start with 1 molecule of a 1000 bp template, 50% GC 1 kb = 2000 nucleotides So , ...


7

Note: In your PCR program you always set extension time. Case: Product length = 500bp PCR extension time = 50sec Assuming that polymerase adds 1000 nt/min Cycle 1: Strand that binds FP: extends ~800nt to the right (as per the polymerization rate): 300 bp ahead of RP complementary site. This product is lets say P1 Strand that binds RP: extends ~800nt to ...


7

I haven't done this exact experiment. I am just deducing from the known facts about PCR. The product of PF and PR2 will serve as a template for both 1kb product (P1) and 2kb product (P2). Lets assume that after 2nd cycle there is 1 copy each of P1 and P2 (Delay of 1 cycle to make a smaller length product. See here). Lets assume that the primer binding ...


7

Melanin is a potent inhibitor of PCR - when you use B16 cells (or any other cell line that produces melanin) you have to purify your sample from it. Unfortunately a simple Phenol-Chloroform extraction or an ethanol precipation won't do the magic, since melanin co-precipitates with the nucleic acids. I recommend following the following paper: "CTAB–Urea ...


7

Short answer All of your sources are correct as they are not mutually exclusive. PCR is used to isolate and amplify DNA to yield small quantities of pure target product. Gene cloning can subsequently be applied to scale the production of the fragment up. PCR thus can be part of the DNA cloning process. Background Cloning in general simply means duplicating....


6

What I can quickly think of is that you were running the gel incorrectly: instead of from - to + direction, You ran it the other way around and your DNA went out of the gel. Another thing might be that the UV light was not turned on or is broken.


6

An expected efficiency for a typical PCR is 80%, meaning each cycle multiplies the copy number of the targeted DNA sequence 1.58 times. Firstly, it makes more sense to refer to the amount of DNA in a polymerase chain reaction in terms of copy number or in terms of moles; the number of DNA molecules of interest is what the reaction is operating on, and the ...


5

You should try another housekeeping gene like GAPDH since the very definition of these genes is that they shouldn't change with your treatment


5

There is one simple reason for that, your agarose gel is most likely too dense. Depending in the type of agarose, I would prepare a 0.5-0.6% gel at maximum. Synbio gives this list for "standard" agarose, which fits pretty good with my experiences. If you use low melting agarose, this table looks a bit different, as the gel matrix is not a dense. The ...


5

No, this does not happen, as the DNA polymerases used for PCR are DNA dependent. This means that they only synthesize DNA when it is bound to DNA. Even if your primers bind to the RNA, the polymerase will not starting new strands here. To use RNA as a template for PCR you first need to reverse transcribe it.


5

It's actually very simple. You misunderstood the term polymorph in this situation. A microsatellite is a sequence composed of a short repeated DNA motif. A polymorph satellite is then simply a microsatellite varying in length between individuals, i.e. composed of more or less repeats of the motif. The flanks of the microsatellite are the DNA sequences ...


5

There are many methods of DNA isolation, so it is difficult to make broad statements about all situations. Typically, though, during the DNA isolation protocol, essentially all proteins are denatured and removed, as is RNA, cell membrane components, extracellular matrix, etc. If you are using a high-quality well-validated kit from a reputable supplier, your ...


5

Edited after clarifications in question, Let's start with normal functions of both enzymes. Helicases separates DNA strands while polymerase synthesize DNA strands as shown in the following figure. (Image Source: Wikimedia Commons) Watch this animation, it will clear your doubts about function. However, RNA polymerase does have both activities. In cells, ...


5

I don't have much expertise in PCR (so please do not ask me technicalities). A research has proven that melanin is a PCR inhibitor as it forms complexes with thermostable DNA polymerase and inhibits its activity, thus inhibiting PCR. See this paragraph below: We found that both RNA and cDNA preparations derived from melanocytes contain a RT-PCR inhibitor ...


5

The reaction for the Sanger sequencing reacting contains beneath the normal dNTP (which are needed to make most of the DNA) the dideoxynucleotides, which lack the 3'-OH-Group and which are labelled with a fluorescent dye. Once one of this nucleotides is incorporated, the chain reaction stops here, leading to a product with a specific length and a known (by ...


5

Short answer: You need to buy some more, but you need the sequence also for ordering. Long answer: The Taq polymerase needs a piece of DNA (or RNA) to prime the reaction and be able to enlarge the DNA chain, this is why we use primers in the first place (also to ensure reaction specificity to the region we want to amplify). To enable the reaction you would ...


5

This is a question I also remember wondering about when I was younger in school. Now as a professional it's way too obvious to even explain. But i think it's an important and common question, which warrants an example or two from common daily lab practice. Preface You have to understand that DNA is a molecule. It's really tiny. It's not trivial to work ...


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 ...


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.


4

OK, let us start from the beginning. We know what makes one cell type different from another cell type is its expression - i.e. what genes are actually being transcribed into RNA. Therefore, there should be certain RNA in one cell type that should not exit in another cell type. If you isolate RNA from a certain cell type, say a neuron and you want to show ...


4

First step is the calculation of efficiency, denoted by lets say $E_{gene}$. See this post for calculation of primer efficiency. So the fold change for that gene will be calculated by $E_{gene}^{-\Delta Ct_{gene}}$ Where: $\Delta Ct = Ct^{treated} -Ct^{control}$ But these Ct values are not normalized. For normalization, you take some reference gene ...


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