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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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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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1answer
20 views

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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17 views

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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22 views

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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19 views

Primer proportion in multiplex PCR

I am just reading a paper about a multiplex PCR protocol. There are 5 primers for 3 targets (let's say primer 1,2,3,4,5 for targets A,B and C). Primer 5 serves for 2 targets. The reaction mix ...
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2answers
38 views

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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36 views

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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19 views

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
34 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
62 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
26 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
49 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
36 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
32 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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17 views

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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1answer
40 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
18 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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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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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
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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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1answer
60 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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0answers
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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
53 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
272 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
19 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
609 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
56 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
110 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
82 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
120 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
89 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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205 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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275 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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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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2answers
63 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
81 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
465 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
38 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
173 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
85 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
78 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
62 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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1answer
102 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 ...