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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

All sequencing methods, be it classical Sanger sequencing or next-generation sequencing (or even third generation) need a certain amount of DNA to work with. You either need to extract DNA from a large-ish tissue sample or you need to amplify DNA content from a smaller sample. The first approach is often impractical, or downright impossible (when you want ...


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

Western Blot tests on young children are practically useless, since they test for antibodies. The child will likely have antibodies passed down by the HIV+ mother, regardless of whether the child has HIV. The test will show the antibodies, which may be mistaken for an active immune response from the child. As such, there will be a high false-positive rate on ...


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

According to their website New England Biolabs use a version of the approach pioneered by Wayne Barnes, as described in: Kermekchiev, M.B., Tzekov, A and Barnes, W.M. (2003) Nucl. Acids Res. 31, 6139–6147 This is basically an assay for the mutation rate in a PCR-amplified lacZ (β-galactosidase) gene, assayed by transforming E. coli, plating on 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

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 ...


4

RNAses are enzymes, and there are various ways to inactivate them. Unfortunately, RNAses are rather stable proteins and autoclaving doesn't completely kill their activity. The common methods to inactivate are unspecific methods that will destroy any enzymes, either through covalent modification or degradation. The most common methods for RNAse inactivation ...


4

Everything depends upon the infection and on the general immune status of the patient. Generally, the prerequisite for DNA to freely circulate in the blood is the presence of bacteria themselves in the blood (bacteraemia). This means that the infection left its original site (where it is usually kept isolated from the blood flow by the immune system). ...


4

We do it all the time. You can use one of your end/flanking primers and use that as a primer for sequencing. Companies will typically have a setup where they can take a "premixed" sample. Since we tend to use sequetech, here are their details: http://www.sequetech.com/requirements.php?premixed=1 For a second opinion look at Elimbio's: http://www.elimbio....


4

Valid XHTML http://nar.oxfordjournals.org/content/29/17/e88/F1.large.jpg. Figure 1. Schematic presentation of the tetra-primer ARMS-PCR method. The single nucleotide polymorphism used here as an example is a G→A substitution, but the method can be used to type other types of single base substitutions. Two allele-specific amplicons are generated using two ...


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