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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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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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28 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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39 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
19 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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28 views

PCR with multiple primers (at same locus)

Does anyone know if its possible to perform a PCR with multiple primers in one run? Usually I have to do two PCRs where the product of the first PCR is used as a template of the second. I couldn't ...
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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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MiniPCR or Biomeme?

I am looking to purchase a handheld PCR device to use in-field for pathogen detection. Trying to decide whether to go with the MiniPCR or Biomeme - does anyone here have any experience with either, ...
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1answer
35 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
31 views

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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Housekeeping and stably expressed genes qPCR

I am trying to find the most stable reference genes in order to use in qPCR assays. For this I picked 2 genes from RNA seq data and 2 genes EF and GAPDH. From these 4 genes I want to pick two genes ...
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35 views

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
15 views

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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27 views

pcr troubleshoot for toxoplasma with GRA6

I did pcr for Toxoplasma genotyping with GRA6 gene. my primer sequences were forward 5-GTAGCGTGCTTGTTGGCGAC-3 reverse 5-TACAAGACATAGAGTGCCCC-3. PCR was done in a 25μl reaction mixture. I used 10pmol ...
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24 views

Gibson Assembly Primers

exchange biologists, Currently having some trouble getting a Gibson assembly to work and as it's my first time working with this method I thought I'd ask for some advice. See the bottom of this post ...
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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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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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Quantifying loss of mtDNA using genotyping arrays

I recently read that mitochondrial DNA (mtDNA) levels can be measured by relative real-time PCR. Would it also be possible to estimate the degree of loss of mtDNA from genotyping array data?
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51 views

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
50 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
204 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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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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1answer
486 views

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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50 views

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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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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47 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
101 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
86 views

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 ...
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1answer
182 views

PCR efficiency or DNA yield with single primer

How to calculate number of DNA molecules synthesized after 'n' no. cycles with single primer (either only forward primer or reverse primer)? Is there any formula to calculate the DNA yield after such ...
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Adjust amplicon size with universal primers

I would like to create primers with universal primers M13. 5'-(Universal seq) - (Adjust) - (Specific Primer)-3' What sequence do I need to (Adjust) primer ...
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248 views

One-sample t-Test for mean fold change

I want to see if there is down-regulation of some hypermethylated genes. I have the mean fold change values from qPCR and have decided to use the one-sample t-Test to see if the changes are ...
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Real time PCR for amplification of long amplicon

As far as I know, the ideal PCR product (amplicon) length should be ranged 100-300bp for doing real-time PCR. However, I got some primers that will be generated products in 900bp and 1500 bp, I want ...
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In PCR what is the chemical makeup of the primer? DNA or RNA? [closed]

I'm thinking the answer is RNA. Is that right?
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PCR gene cloning

I've been set some homework and I have no idea where I would start. Any help would be appreciated. Thanks Which version of primer 1 should be used to allow the maf gene to be cloned into the pQE-30Xa ...
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How to conclude my qPCR analysis? (Eggplant-drought stress)

I did an experiment with two varieties of eggplant. The plants were subjected to 3 weeks of water stress and after the experiment we performed qPCR. We used the a gene which encodes a NAC ...
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59 views

Can PCR duplicates have complementary sequences?

It is common when analyzing paired-end whole-genome shotgun sequencing data to check for and eliminate PCR duplicates. The reasoning is that the probability of sampling a fragment of the genome of the ...
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1answer
76 views

Interpreting qPCR curves: how to find Ct value in my case?

I am reading my qPCR results and having no idea why Ct value is returned like this: This is the qPCR curve of my samples, and the software returned Ct values of most of the curves as seen: 33.99-38.4 ...
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5answers
415 views

PCR products with no band in gel

I am a PhD student and stuck with the no band in the gel results. I am using the following 1 microliter Primer R (20mM) 1 microliter Primer f (20mM) 1 microliter dNTP (10mM) 1 microliter DNA ...
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2answers
36 views

Does PCR amplification affect microarray or DNA biosensor results?

I've noticed that PCR amplification is commonly done before analyzing the DNA sample with microarrays or biosensors. However, won't amplification biases or errors mess up the gene expression profile ...
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1answer
158 views

How can the melting temperature of PCR primers be so far below the extension temperature?

Since our primer TM is lower than the extension temperature by design, (and often by a significant amount) shouldn't the primers be released from the template at extension temperatures? Now, I have ...
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1answer
78 views

PCR related problem

Recently, I have done a PCR of a region of human p53 gene and got faint band after that. Then I used that PCR product as input and did a nested PCR with same PCR condition, but now the specific band ...
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1answer
76 views

How does a polymerized TruSeq adapter affect sequencing reads?

I am trying to build a NGS library on enriched DNA (DNA length = 93 bp). I am using PCR amplification with two overhanging primers, both containing Illumina adapter sequences (Universal Adapter & ...
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1answer
57 views

How is an RT-PCR representative of the RNA contents in a cell?

For example, we first collect all the RNA contents from a mammalian cell line. Say, for instance, we collect 80 uL of RNA with a concentration of 150 ng/uL. Next, to make cDNA, we take 1,000 ng of the ...
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96 views

Can qPCR primer with several mismatch works?

I am designing primers for qPCR from conserved regions of a few different fish species.This is because gene sequence for my fish species are yet unavailable in NCBI database. Is it OK (can the primer ...
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1answer
90 views

Is it possible to produce a tangible mass of DNA/RNA using a PCR reaction?

A Polymerase Chain Reaction (PCR) is a method of amplification which enables one to produce many copies of a certain DNA/RNA strand for many applications (as described in the link above). I have ...
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1answer
4k views

Why is only one primer used for DNA sequencing instead of two?

I understand that PCR uses two primers that anneal to the two ssDNA's in order to exponentially amplify a DNA and that Sanger sequencing uses only one primer because a sequence can be determined with ...
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1answer
634 views

Questions regarding PCR

Background: From Genomes: The temperature of a PCR mixture is first set at 94 °C so that denaturation of DNA can happen. Then it is reduced to 50–60 °C, which results in some rejoining of the single ...
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206 views

Is PCR a DNA cloning technique?

According to Genomes PCR is A technique that results in exponential amplification of a selected region of a DNA molecule [in test tube]. DNA cloning is Insertion of a fragment of DNA into a ...