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11

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 ...


11

(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 ...


8

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 ...


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

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

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 ...


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 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 ...


6

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 ...


6

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 ...


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

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 ...


6

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 ...


5

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 ...


5

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 , ...


5

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.


4

This paper describes some PCR strategies with LINE and SINE PCR identification (Shedlock and Okada. SINE Insertions: powerful tools for molecular systematics. BioEssays (2000) 22:148-160.). I have no experience with PCR amplification of SINEs or LINEs, however I can think of two strategies right now. 1) You may be able to find a unique 18-20 nt region ...


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: ...


4

There is no single "ideal amount" of RNA. I would suggest you to do a titration curve to determine the best amount for your specific assay. The band at 100 bp could be a non-specific amplification. You could try to increase slightly the annealing temp to see if that removes (or reduces) the lower band. Alternatively, you can consider a different primer set ...


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

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 ...


4

. 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 pairs of primers, one pair (indicated by pink and red arrows, respectively) ...


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 ...


3

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). ...


3

Please provide more information: fold-change relative to what? If you did what I think you did (single control gene that you calculated fold change to of your gene of interest) I'd say this is the wrong approach. What you need is a set of genes which have similar expression levels across all your samples (controls and cases) to be able to compare your gene ...


3

In my own personal lab experience, I got some unexpected results using Phusion polymerase. However, I have not seen deletions, only insertions (ranging from single bases to 3 tandem copies of a primer sequence). This is not an answer to your deletion question, but it does suggest unusual things might happen. I have seen deletions with T4 DNA polymerase ...


3

I've run a test following this protocol to the letter... PCR step by step: Collect all ingredients (excluding TAQ) from the refrigerator and keep cool (on ice). Leave the TAQ in the freezer until required. Make master mix by adding all ingredients (excluding TAQ) using fresh pipette tips for each ingredient and using the volume specified in ...


3

Genomic DNA is a DNA that has whole set of genetic data of an organism. You usually extract it with a whole DNA extraction kit. Vector DNA is a vector (usually a plasmid, it can also be artificial chromosome,...the type of vector depends on the species and strategy you are using) that has you desired string of DNA in it (for example a gene you want to ...


3

We used this kind of primers to generate out of frame mutations or to add additional bases. In my experience your PCR will work (probably a a lower efficiency) and you will get a product with an additional base. We used primers with bigger differences in PCR based site directed mutagenesis of plasmids, there up to 10 bases didn't match but the primers where ...



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