Hot answers tagged

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

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

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

It is actually good if both the primers have the same Tm (Melting Temperature) because you would basically want both primers to anneal at your Ta (Annealing Temperature) which is generally set as Tm-5. If the Tm(s) don't match, then your Ta should be set according to the lower Tm.


4

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 ...


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

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

Although this is theoretical a problem, in fact it isn't one. Lets do the calculation around it. EDTA complexes equimolar amounts of bivalent cations (e.g. Magnesium), so 1mM EDTA will complex 1mM Magnesium ions. Lets assume that the PCR reaction contains 2mM Magnesium. A typical PCR reaction I do has a total volume of 25ul and contains 1ul DNA template in ...


3

The References and External Links sections of the Wikipedia article in your question contain a great deal of relevant information to get you started. There is a link to the FBI's CODIS and NDIS (National DNA Index System) Fact Sheet, which contains a lot of relevant information, including part numbers for validated kits from several manufacturers. DOI ...


3

The DNA polymerase also needs a RNA primer on the leading strand to be able to start polymerization. Afterwards this is not needed anymore, since the replication goes on without a break. On the lagging strand polymerization replication can only work between the replication fork and the next region of double-stranded DNA. See the figure (from here): The ...


3

Not a stupid question at all. I was just as confused by this as a beginner in molecular biology. You get the hang of it after a while. :) I find it much more difficult to design primers from just one strand, especially the minus strand, so I always try to get both to work with. If you don't feel like doing that, just one strand is sufficient (plus or minus),...


3

This dates back to the 1980's when these M13 primers were first cloned. Heidecker G, Messing J, Gronenborn B. 1980. A versatile primer for DNA sequencing in the M13mp2 cloning system. Gene 10(1):69-73. In this paper, they discuss the construction of a primer for sequencing of the M13mp2 phage vector. An EcoRI/AluI digest releases a 92 bp fragment from a ...


3

The leading strand post-initiation First consider the DNA replication fork after initiation has occurred. The 3′-OH primer on the leading strand is the 3′-end of the strand of DNA being synthesized in the 5′ to 3′ direction (circled in the diagram below, modified from Berg, Biochemistry). It is not removed because there is no need to do so. (The primer on ...


3

The default parameter provided by "primer-blast" is 3 degree (max difference between the melting temperatures of the pairs) . that said I think it really depends on the application. in case of cloning (in my experience) you can allow wider ranges of delta Tm between primers (in this case I used to use the average Tm of the two primers as annealing ...


3

This really depends on your application, but from my experience 2-3 degrees with a max of 5°C difference usually works. The more the melting temperature differ, the more you need to optimize all the other parameters like salt and magnesium concentration, amount of enzyme, annealing and elongation times and so on. 85°C melting temperature seems very much for ...


3

A few things to consider: Template: Are you sure the template you want is on a plasmid, and not in the Rhizobium genomic DNA? (In which case it would not be present in DNA purified with a plasmid miniprep kit - you would need to do some kind of genomic DNA prep instead.) Have you measured the concentration of your template DNA? I typically put ~2-10 ng of ...


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

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 ...


2

Lets summarize the comments into a real answer: When the only chemical changed in your reaction is the DNA, which came from a fresh Phenol/Chloroform preparation, I would suspect it. From there, we have a few possibilities, what could have gone wrong. The most likely cause of an enzymatic reaction not working with Phenol/Chloroform prepared DNA are left-...


2

First of all, if this is a homework question, add that tag to it. And show what work you have done so far. Secondly, primers for amplifications should lie on opposite strands. Primers are typed in 5'-to-3' direction (aka left-to-right on leading strand). Appropriate primers will be: primer 4=GTG... and primer 5=GAA.... Note how those primers are always in ...


2

If there is a such a site, I have never encountered it. Since you say you have the genomic coordinates for each primer it seems to me like all you need is a list of the genomic coordinates of every transcribed exon in the human genome. Assuming that is correct then you are in luck because such lists do exist in the form of a GFF3 file. You should be able to ...


2

I think various ways can be apply, but the easiest way could be to indicate the range to set a forward primer. Decide the length of primer you are going to get. Let's say you would like 25 mer. You can set the range from chr12:25398261 to chr12:25398309. When you identify where chr12:25398285 is in NG_007524.1, you are ready to design your primers.


2

You can have GC-clamps (3' terminal G/C) in both primers; this may overall improve the PCR efficiency. However you should take care not to put too many G/Cs. This may lead to stabilization of primer pairs, leading to formation of primer-dimers. Also see this post: When designing primers how important is the GC clamp? GC-clamps are not always essential and ...


2

The conventional Eiken LAMP formulation has an excess of Mg2+. 5 µl of TE with EDTA at 1 mM going into a 25 µl reaction will have minimal impact. In any case the LAMP formulation can be up adjusted in Mg2+ to compensate, but I doubt this is even necessary. Besides, bugs will lyse better in the boiling method with EDTA present and it will hinder nucleases.


2

As you correctly point out, designing an optimal primer pair for 16S-rRNA sequencing is a tricky affair because even the less-variable regions are not same between different strains and species. Sambo et al (2018) have even developed a bioinformatics software for optimal design of primers for 16S-rRNA sequencing for multiple bacteria. We propose here a ...


2

If you are not getting amplification, there are multiple things to visit before you want to design internal primers. You should try troubleshooting your PCR reactions before designing and ordering internal primers. If you are doing the PCRs wrong, it is likely you will run into the same problems again with internal primers. There are excellent guides to ...


2

From the description, yes, those primers are supposed to be specific for the IL-2 gene. The way they would be used would be on mRNA, not DNA, so you could use them to quantify the level of IL-2 transcription. However, there seems to be some glitch in there, because while the first sequence (5′-CAGCTGTTGCT GGACTTACAGG-3′) matches rat IL-2 mRNA starting at 120 ...


2

The very extremities of sequencing reads obtained by most if not all sequencing technologies are usually of lower quality, though more often so in the 5' region. You should disregard this data, or better yet design additional primers further away to encapsulate that region too if you desperately need it. Below is a fairly typical output from FASTQC analysis ...


Only top voted, non community-wiki answers of a minimum length are eligible