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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 ...


9

Perhaps you can draw inspiration from classic paper on lambda cloning: Maniatis T, Hardison RC, Lacy E, Lauer J, O’Connell C, Quon D, Sim GK, Efstratiadis A. 1978. The isolation of structural genes from libraries of eucaryotic DNA. Cell 15: 687–701. Try selecting tissues from the animal which you think is "enriched" (i.e. highly expressed) for the specific ...


8

Your teacher is indeed correct. In the first round you would get two identical molecules of the dsDNA. In the second round you would get 3 identical molecules and one molecular with an A substituted for a G in one of the strands. ie. No error (3 of the 4 molecules): ------G------- ------C------- One mismatch (1 of the 4 molecules): ------A------- -----...


8

Deletions may make sense if you are analyzing the N-terminus or C-terminus of a protein. If you are looking at an internal region however, keep in mind that the more AAs you delete, the more likely you are to disrupt the overall protein structure. If you delete any random selection of 8 AAs within a protein, there's a chance you'll knock out activity by ...


8

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

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

While it isn't the cheapest, it is certainly the fastest and simplest. I would quikchange out the amino acid. This would require no subcloning and only require two ~25 nt primers ($10) 1 shot of pfu (~$0.25) 1 shot of DPNI ($0.05) competent cells (~$5) sequencing to confirm (~$4-6) Overall, probably >$20 a mutant all in 2-3 days of waiting. (edit) I'm ...


7

I will format it to .ppt as soon as I have more time! If something is not readable, please let me know! I have to point out several things: The fraction of incorrect DNA molecules does not depend on the number of cycles (as long as the number of cycles is higher than 2). There is one exception: if the mutation occurs outside of the region to be amplified (...


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

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

Primer Tm calculations can vary significantly based on the method used. What I can tell you, is that the Tm really depend on the polymerase you are using for the PCR reaction and for each polymerase there is a set of PCR conditions you have to follow. As I use Phusion® Hot Start High-Fidelity DNA Polymerase from Finnzymes, I will give you an example with it:...


6

I don't think primer dimers are your primary concern here. Usually in my experiences, I get primer dimers all the time, even if the reaction works and I get my bands of interest. Maybe you ought to troubleshoot other aspects of your PCR that might account for why your reaction isn't working. Have you tried using a positive control with your primers? You may ...


6

I see big fuzzy bands around 100bp as well. They're most likely RNA contamination. To get rid of them, digest your RT-PCR products with RNAse-H. But if you just need to visualize your band of interest, and the fuzzy bands aren't getting in the way, it shouldn't be a problem. I usually input anywhere from 1-2 ug of RNA into my RT-PCR reaction using the ...


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 ...


6

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

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

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, ...


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