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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1answer
857 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
708 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
42 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
121 views

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
63 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 1 ul of my template last into the 96 well plates (on ...
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1answer
35 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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1answer
70 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
21 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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2answers
24 views

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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321 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
120 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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20 views

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
62 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
537 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
23 views

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
2k 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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1answer
106 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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3answers
189 views

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
158 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
281 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
100 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
303 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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2k views

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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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
146 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
680 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
44 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
217 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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98 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
83 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
69 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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119 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
153 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
8k 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
1k 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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279 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 ...
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1answer
38 views

Can conventional PCR amplify DNA from different organisms from a specimen in a single step?

I've understood so far that in conventional PCR, the most abundant DNA/genotype present in the speciment at the beginning of the reaction is selected and esponentially amplified. So that cPCR are ...
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1answer
95 views

Meaning of “primers IL-2” in a scientific article

From an article ("Amelioration of Experimental Autoimmune Encephalomyelitis in Lewis Rats by FTY720 Treatment", 2003): We performed PCR amplification in a 100-μl reaction mixture containing 200 μM ...
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Trouble amplifying microsatellites using a 3 primer protocol, positives in negative controls

I'm trying to collect microsatellite data using primers developed on a related species but instead of having primers with fluorescent dye already on them, I'm trying to attach an M13-dye to the ...
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1answer
11k views

Manual Primer Design for a gene on the reverse strand

My question might sound very naive and stupid but I am hopeless now. I read so many websites and pages but could not figure out this PCR primer design thing completely. Some genes are on the reverse ...
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517 views

What's the best wat to combine biological replicates in qPCR gene expression analysis? (2^(-∆∆Ct) method; one-way anova, turkey's test)

I am measuring the levels of gene x after transfection of cells with a plasmid and I want to compare them to the levels in mock-transfected cells. I did three different transfection experiments, in ...
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2answers
1k views

Why should you use an annealing temperature about 5°C below the Tm of your primers?

Why should you use an annealing temperature about 5°C below the Tm of your primers? According to my current research, I think it has something to do with the other reactents in the PCR, but I am not ...
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1answer
6k views

Maximum loading volume for agarose gel with 10 well comb

I want to load my agarose gel with my PCR products, which are 50 µL reactions. I am wondering how much of those reaction I can load into one well if I use a standard agarose mini gel (10 well comb, ~...
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2k views

Designing primers with restriction sites

I want to design a forward and reverse primer that include overhangs with restriction sites, with the dna used for restriction enzyme cloning. If I want to send my dna to a synthesis company should ...
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2answers
68 views

Performing PCR on samples whose DNA concentration is nul

Is it vain to perform a PCR on samples whose DNA concentration was measured as nul by a NanoDrop?
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1answer
181 views

RT-qPCR for RNA quantification

I'm really new to RT-qPCR and probably I've just a minor problem than I think. So, what's my problem about. I want to quantify a virus by a specific area within a gene on its genome. I've specific ...
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1answer
205 views

What is a PCR test?

I know that PCR is used to amplify DNA, but what do people mean when they mention 'PCR tests' for diagnosis of disease? And what does a positive/negative PCR test mean?