Questions tagged [pcr]

Polymerase Chain Reaction is a method of replicating DNA exponentially from even a single molecule.

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Why is my DNA band bulging?

This is the only image my the TA was able to get for us. And, we're using it for our lab report. The image isn't even ours. It's another group's image that we're sharing. But I don't understand why in ...
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Is there a biological or technical reason for a 40 Ct cutoff in nucleic acid detection assay?

I have developed and validated a modified a nucleic acid test (NAT) for SARS-COV-2 detection using real-time RT-PCR (aka rRT-PCR, aka RT-qPCR). My assay is not for diagnostic use, but for donor ...
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Polymerase Chain Reaction Specifics

While going over PCR in my biology lecture this week I have come across a few questions I have about this process. First, since PCR focuses on trying to replicate a specific targeted DNA sequence many ...
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LAMP (isothermal amplification), hydroxy naphtol blue , disodium salt" or just hydroxy naphtol blue?

I am reproducing this work on LAMP (isothermal amplification) Article A new visually improved and sensitive loop mediated isotherm... , but I am a bit confused about the difference between "...
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Why PCR primers don't amplify beyond the target region? [closed]

I have been doing PCR for a while. I spared a bit of time to look it up but could not find an answer. So, for sanger sequencing, if we have a primer, it can amplify about 800-1000 bp. But, if you ...
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RT-PCR: Not seeing how it can measure mRNA expression levels

From my understanding, in RT-PCR we start off with an mRNA molecule, use the enzyme reverse transcriptase to create a cDNA copy (hence creating a double-stranded mRNA / cDNA hybrid), degrade the mRNA, ...
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Meaning of “standard reactions” in a DNA extraction procedure description

From a DNA extraction procedure description (an in-house pharma document I'm translating into Russian): Preparation of Standards All the standard reactions should be prepared at least in duplicates. ...
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Meaning of “optical” in “Optical cap, 8X Strip” (used in qPCR)

I'm translating an English document that lists equipment used in a qPCR procedure: Reagents/Materials Optical cap, 8X Strip I googled and found that the meaning of this line is "a strip of 8 ...
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Construct aptamer library with exact number of members

I am asked to construct an aptamer library of exactly 1024 members, each with a unique V. I have two questions regarding this; firstly, is it wrong to construct an aptamer library using a column with ...
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High concentration of primer in PCR

Why is it that higher concentration of primers than the DNA template in PCR will favor the annealing of a template to a primer?
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Humic Acid and PCR

Quite a few papers claim humic acid is an inhibitor of PCR reactions. I understand this is true when working with soil microbes, but how does it qualify to be a PCR inhibitor in general (i.e when not ...
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Are specific primers or detectors, or both, used in COVID-19 tests?

I am trying to learn about the rRT-PCR testing procedure used to test for COVID-19, but I am slightly confused on one point. Are highly specific primers used with a non-specific detector, or are ...
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Is there a difference between a consensus PCR and a pan PCR?

I know that a multiplex PCR uses multiple sets of primers in one reaction to amplify multiple targets. But what is a PCR called that uses generic primers to amplify multiple targets? Is this a ...
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Why there is no ELISA for SARS-CoV-2?

As far as I know, SARS-CoV-2 tests currently used worldwide are real-time RT-PCR. Why there is no ELISA for SARS-CoV-2? Compared to PCRs, ELISA is: Way cheaper; Way faster; Does not require trained ...
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What are the trade-offs between polymerase chain reaction (PCR) vs Loop-mediated isothermal amplification (LAMP)?

I've heard that LAMP could be a good alternative to PCR for DNA strand amplification and detection because the equipment required is cheaper, more portable, and the amplification is faster since all ...
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How to recalibrate qpCR reading when incorrect qty of solution is added during DNA elution step?

I have 20 viral DNA samples collected for 6 time points. The DNA content varies over time My DNA extraction protocol step suggests that in the last step of the protocol during elution I had 100uL of ...
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Swing bucket for PCR

I see a swing bucket rotor for PCR trips and don't know what step is using? What difference if I use fixed - angle? In addition, Which step I can use swing bucket instead fix - angle in molecular ...
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How to calculate the quantification of qPCR to the equivalent of the number of nuclei in fungi?

I have a question about the quantification through qPCR. The my question is: if I made the qPCR of a fungal functional gene, how is possible to obtain from the quantification number (in nanogram) the ...
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How to design the primer when you don't have GC?

I have treated my RNA with sodium bisulfite so all my cytosine is converted into thymine. Then I used that RNA for my RT reaction. I have a cDNA template without Cs. Now I am trying to amplify that ...
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How long does the overlap have to be to reliably join two DNA strands with overlap PCR?

I was having a look at how Overlap extension PCR as part of a de-novo DNA synthesis mechanism. I was thinking that first smaller pieces would be synthesized somehow with overlaps, and then joined ...
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PCR markers for C57B6

Do you know any primers that can be used to genotype mice and check if they are still C57B6? I'm concerned about genetic drift in my colony. I bought a breeding pair 3 years ago and expanded. I wanna ...
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Minimum amount of RNA for cDNA conversion and qPCR

I searched several protocols for reverse transcription of RNA and all suggest using RNA concentrations of 1ug. Could a lower amount result in inferior transcript representation and therefore decreased ...
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Taq Polymerase's stability at high temperature

I was asked a question as to what is so special with Taq Polymerase that makes it quite stable at high temperatures though its functioning is the same as other DNA polymerases like that of mesophiles. ...
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Is chromosomal DNA more likely to interfere with restriction mapping or PCR analysis in E. coli?

We are characterizing YADH-1 in my biochemistry lab course. Over the course of the first weeks, we harvested E. coli cells, and isolated plasmid DNA via alkaline cell lysis. A previous exam for the ...
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What would be the effect of excess taq polymerase on the PCR?

I just had one question regarding the possible effect of putting to much Taq polymerase in my PCR tube? Instead of 5µl I put 50µl (10x more). Do you think it will have a bad effect on the reaction??...
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Why do we need to amplify DNA sequences?

I am learning about biotechnology. I have no education in biology or chemistry and am simply interested in the subject of biotechnology. I am wondering why we need to have multiple copies of a piece ...
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172 views

Can primers for PCR be duplicated?

Complete beginner question here, don't laugh: If I have some primers that have been synthesized, and I am close to running out of them, is there any way to duplicate them / amplify them / synthesize ...
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Why is the Tm defined as the temperature at which 50% of dsDNA has changed into ssDNA?

In molecular biology, Tm is defined as the temperature at which 50% of dsDNA is converted to its single stranded form. Intuitively it would seem that the melting temperature should have been defined ...
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What does the final (Illumina) RNA-seq library contain? ss cdNA or ds cDNA?

I guess the oligos on Illumina's flow cell only hybridize to to single-stranded cDNAs (sscDNA). However, when I read the TruSeq protocol (googled: TruSeq® RNASample Preparation v2 Guide) at the PCR ...
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Why does one combine PCR and cloning as ways for amplification of sequences?

Why does one combine PCR and cloning as ways for amplification of sequences? Don't they produce the same result? I was reading the paper https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1864885/ and got ...
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How do primers anneal to ssDNA?

In a PCR type protocol, the high temperature stage of the cycle will cause dsDNA to denature, as well as any annealed primers. At the lower temperature the denatured dsDNA will remain denatured. As ...
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In touchdown PCR, should the extension temperature match the annealing temperature in phase 1?

For TD PCR, it's recommended that the annealing temperature (T_a) be 10°C higher than the normal cycling T_a for the first 10 cycles or so, dropping 0.5 to 1.0°C/cycle. Should the extension ...
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I did an agar gel electrophoresis but I could not see DNA, why? [closed]

After performing a PCR, I ran the products (700 bp) in an agar gel electrophoresis along with a Genedirex ladder (100 bp), but the transilluminator did not reveal them.
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PCR in different cell line

I developed a PCR cycle to amplify a 5.5 kb genomic DNA region in HEK cell line. When I tried to use the same protocol in A375 cell line I had no results but only smear. Can you help me? Thanks in ...
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Issues with Mutant Allele Fraction Preparation

I am currently trying to prepare mutant allele fraction (MAF) solutions for an allele discrimination qPCR assay. I began with plasmids cloned with the different desired alleles. I amplified the the ...
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Gene cluster of interest not being amplified in PCR

In a lab, I currently have a sample of Rhizobium sp. NT-26. This bacteria is a chemolithoautotrophic arsenite-oxidizer, and I want to clone the arsenite oxidase genes into another bacteria strain in ...
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Can Dpn1 digest hybrid PCR product with only one methylated strand?

In Site directed mutagenesis using PCR, after a cycle we obtain a hybrid molecule with one parental strand and other newly synthesized unmethylated strand. This is followed by Dpn1 digestion. Does ...
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Why both forward and reverse primer are added in asymmetric PCR?

If the purpose of Asymmetric PCR is to amplify single targeted strand of dsDNA then instead of adding just one primer for targeted strand why do we add both forward and reverse primer? Upon addition ...
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Can PCR detect phosphorylated protein? [closed]

I'm trying to detect the level transcriptional protein inside cells. However, the cells contained both of this protein that is phosphorylated and not phosphorylated. Can RT-PCR detected the level of ...
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PCR Mastermix Preparation

I am currently preparing a PCR mastermix for the first time, and have a few questions regarding the distribution. I am trying to amplify a specific gene cluster in a sample of DNA so I can use it in a ...
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Trying to find the right plasmid expression vector for e. coli c600

I am very new to the concept of gene cloning and transformation. I am trying transfer the arenite oxidase gene cluster (9kb in length) to E. coli strain C600. I want the E. coli strain to express this ...
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Maximum length for PCR amplification

Is there a recommendation for how long a target sequence for PCR amplification should be? If there is how do you amplify a target sequence that exceeds the recommended length?
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How to distinguish the Ct of the target gene from the Ct of the housekeeping gene? (very basic qPCR question)

I will do a qPCR experiment for the first time, and I have a bottom-level question to ask. I know that for normalizing the quantity of your target gene you have to add an endogenous control, like a ...
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How is a specific fragment isolated for PCR amplification?

For background I am interested in studying engineering applications of a specific protein, which is not commercially available. My end goal is to express the gene for the protein in bacterial cells, ...
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Join a linear plasmid just using a primer (without ligase enzyme)

Can I use just a primer and PCR to join the cutted plasmid like this ? without using any further enzyme like ligase. After the linear dsDNA plasmid annealed into 2 ssDNA, primer will bind to middle. ...
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Can we spike with a different enzyme to a SYBR Green Master Mix?

I followed the standard SYBR Green Protocol for doing a qPCR. For which I used 10 uL of 1X SYBR Green Master Mix Forward Primer and Reverse Primer (each at a final conc. = 8.5 uM) Template (unknown ...
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727 views

Calculating PCR Cycles

I have a problem understanding a PCR exercise, it says to calculate the number of cycles to get 1µg of the DNA fragment, starting from 10ng and then from 500ng, I know about 2^n, and the exercise even ...
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Why is DNA polymerase added at the end of PCR reaction?

PCR reaction is used to amplify DNA fragment. Each reaction requires a DNA template, buffer, dNTP mix and a unique pair of primers, one ''forward'' primer and one ''reverse'' primer. The DNA ...
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Primer design for PRP24-TAPtag

I have a problem with my proteomics homework. I was asked to design primers used to create a homologous recombination box to create PRP24-TAPtag yeast. I know that homologous regions should be 40-50nt ...
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too low extention time for Taq polymerase PCR

I am currently working on my masters degree and have recieved the following question: "you want to amplify by PCR a gene 1kb long. The Taq Polymerase you have in the lab is able to synthesize 1kb/...

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