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Questions tagged [pcr]

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

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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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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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1answer
295 views

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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GC Clamp In Primer Design ,, what is the max number of GC Clamp? [duplicate]

I'm Designing a primer and I want to Know what is the Max Number of the GC clamp Could be in the primer? Thanks in advance
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Multiplex PCR question

Multiplex PCR question - I'm trying to understand Multiplex PCR. From watching this video I'm getting the idea that Multiplex PCR isn't quite the the same principle as qPCR. This seemed to be the only ...
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At what point in time is rtPCR (qPCR) no longer quantitative?

Regarding qPCR, after how many reaction cycles typically does this procedure become non-quantitative. In other words, how long does it take for the reaction reagents to fall to too low a level? Thank ...
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1answer
52 views

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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What is the advantages of using single primer in PCR ( Being found in library preparation step of Smart-seq2)

I am curious about the advantages of using single primer in PCR. I found this strategy in single cell sequencing method Smart-seq 2. Will it raise the yield of PCR? If so, Why? Does anybody have ...
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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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2answers
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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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1answer
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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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1answer
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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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Why is snoRNA often used as a reference gene in qPCR?

I am studying non-coding RNAs and stumbled into a question about small non-coding RNAs. scaRNAs (small cajal body-specific RNAs) is a subtype of snoRNAs which are small nucleolar RNAs. It is known to ...
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1answer
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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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1answer
202 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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1answer
375 views

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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1answer
37 views

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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2answers
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How different can the Tm be between 2 primers?

I have a forward primer with Tm 85 degree Celcius. I cannot change this primer. But I have two options for the reverse primer. One with Tm 65 degree Celcius (Annealing at 65 degree Celcius) and ...
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1answer
42 views

qPCR : Cross-contamination while sealing plate

I'm trying to do realtime PCR on a plasmid and I have my positive and negative controls close to each other along with a no-template control. I add 1ul of my template last into the 96 well plates (on ...
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1answer
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Why doesn't my rtPCR reaction work?

I am doing a rtPCR to detect the watermelon mosaic virus (WMV). My set of primers are: WMV primer forward: 5'-TNGARAATTTGGATGYAGG-3' WMV primer reverse: 5'-CTGCGGTGGACCCATACC -3' both of which at ...
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Why is the relative expression in qPCR so low?

When analyzing different mutations through qPCR I found the $2^{-\Delta\Delta ct}$ value for each mutation. All the mutation have similar values compared to the wild type gene, however one of the ...
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1answer
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Is there a biological explanation for a 0.5 difference in allele size with PCR product?

CONTEXT I am currently working on a set of diversity, this diversity in interspecific (within the same genus). I am using SSR markers, the primers were designed on one species and are working really ...
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2answers
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human gene PCR primers database

I wonder if there is a place on the internet to get PCR primers for a specific gene. For instance, exon 15 in CFTR gene. I don't want to design myself because I am sure sombody else already did it.
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2answers
248 views

Why is PCR possible to amplify DNA template before Sanger Sequencing for unknown DNA sequences?

Most of the informations show that both PCR and cloning can be used before Sanger Sequencing to amplify the DNA template. But how is it possible for us to design the primers if we don't know the DNA ...
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1answer
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Which comes first, PCR or Gel electrophoresis?

If I want to find out wither a group of patients have the abnormal BCR-ABL cancer gene, how do I benefit from the PCR and the gel electrophoresis techniques? I'm kinda lost trying to determine which ...
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Minimum number of cycles for effective qPCR quantification?

I have been having an apparent problem with gDNA contamination in my no-reverse transcriptase (NRT) controls by the appearance of fluorescence peaks in my qPCR data. I have/am trying multiple DNase ...
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1answer
57 views

PCR a plasmid with self-complimentary regions?

I am trying to do a PCR using a plasmid as the template, pretty basic stuff. But the problem is that my forward primer just happens to bind to another location on the plasmid in the reverse ...
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1answer
440 views

When using a primer to replicate DNA in a plasmid, does it replicate the whole plasmid?

I've been learning about PCR and plasmids. I understand that the reason for having both a forward and a reverse primer is to extract and amplify the specific piece of DNA between these two primers. ...
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1answer
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In a Sanger sequencing, what would be happen if different DNA sequence binds to same primer? And how this situation avoided if problematic?

Sanger's PCR chain-termination sequencing, by its theory, is a simple method to sequence DNA from organisms. But what will happen if there are 2 allelic DNA with same primer binding site, but ...
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How to linearize target vector by PCR

In my bio class, we learned that you can use restriction enzyme or PCR to linearize a plasmid. Linearizing by restriction enzyme seemed visual and intuitive but how do one linearize a vector using PCR?...
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How long does it take for DNA to degrade

Can i still use 3-4 years old bacterial DNA template for my PCR? They were kept in -20 all this while. Will the DNA degrade?
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3answers
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PCR not working

I am trying to check the integration of DNA into a very specific region of the E. coli genome, and for that I'm comparing via 2.5% agarose gel electrophoresis the length of whole cell PCR products (I'...
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1answer
143 views

double PCR with tailed primer

I would like to make a double PCR with universal tailed primer. The first PCR would make a tailed amplicon. The second PCR would generate fragment labeled with fluorescent dye. Here is my protcole. ...
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1answer
172 views

qPCR (weird ntc curve, amplifies before templates and reaches plateau quickly)

I'm new to qPCR, and I’ve been testing the AR expression on canine tissues. I’m currently using a pair of primers that have been tested in other article for canines.The issue I’m having is that I ...
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2answers
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Dealing with Repetitive Bases in DNA?

I have been given a project involving a plasmid that contains long stretches of Adenine (60 or 120 bases each). These PolyA stretches are interrupted by the occasional G or C. I understand that ...