Questions tagged [pcr]

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

53 questions with no upvoted or accepted answers
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2answers
736 views

How does LCR compare to Assembly PCR

The question pretty much explains itself. How do the two methods compare? I've always used Assembly PCR but the method is prone to mistakes and I'm curious how it compares to Ligase Chain Reaction (...
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0answers
88 views

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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0answers
43 views

Unknown Issue with qPCR Standard

I am currently doing the ground work for a SELEX experiment and I am attempting to verify my standards for the starting library. I ran a reaction with samples containing 50, 5, 0.5, and 0.05ng of my ...
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0answers
39 views

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 ...
3
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0answers
2k views

How to solve the problem of 2 melt peaks in real time PCR?

I am facing a problem in my real time experiment. After standardizing the annealing temperature by semi-quantitative PCR, I did real time for my gene of interest. In one of my samples, I got 2 peaks ...
2
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1answer
88 views

SARS-COV-2 replication speed

What is the speed of replication of SC2? Any information, including in vitro data would be appreciated. I would be interested to know the length of SC2 eclipse period, latent period and something like ...
2
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0answers
34 views

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 ...
2
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0answers
22 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 ...
2
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0answers
34 views

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 ...
2
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0answers
537 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 ...
2
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0answers
71 views

PCR limits of detection calculate method

Is any standard method for use to gain PCR sensivity or limit of detection? my mean is not for RT-PCR or Real-Time or any other kind and just simple PCR. After making PCR product free of dNTP and ...
2
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0answers
289 views

PCR that worked previously is now only showing primer dimers and a smear on gel

PCR amplification of a promoter sequence for gel extraction worked beautifully using Phusion HF enzyme with GC (higher error but less picky) buffer. However, DNA concentration from the gel extraction ...
2
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0answers
59 views

What evidence is there that common PCR DNA purification kits can isolate DNA from pathogens engulfed in macrophages?

I have previously asked a more general question about how PCR sequencing reaches all possible DNA targets here. I want to ask more specifically now: what evidence is there that common PCR DNA ...
2
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0answers
85 views

BSA for trouble shooting failed PCRs?

I am using PCR to amplify a 1000bp region of the CytB mt gene. Some of the samples had not amplified and I wish to change the reaction components (or use additives like BSA) to attempt at getting ...
2
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0answers
17 views

Surface Power Density of Fluorescing Reaction

I am looking to find out what is the ballpark power density of a fluorescing PCR reaction using a SYBR Green or Taq Man dye might be. Is it on the order of $E_e=1mW/cm^2$, less or more? Any help of ...
2
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0answers
61 views

What is the maximal insert length for PCR based homologous recombination in S. cerevisae

I would like to insert a 6 kbp construct, which I have on a plasmid into the genome of S. cerevisiae. This plasmid was originally constructed to integrate at the HIS locus via homologous recombination ...
2
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0answers
101 views

Based on which criteria should i choose the control sample to calculate delta_delta_Ct

I want to calculate the gene expression of an experiment with the Delta delta Ct method. I have these results from two runs of a real time PCR (one for the GOI(Gene of Interest) and one for the ...
2
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0answers
36 views

How closely correlated is the level of an snRNA with the level of its corresponding snRNP?

Let's say I am growing cells under two conditions, and let's say I measure U1 snRNA levels in both using digital PCR. If the U1 snRNA level is reduced in one sample, how safe is it to say that U1 ...
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0answers
46 views

What are the causes of gene amplification in archaea?

I was studying this article about Genome Sequencing of a Genetically Tractable Pyrococcus furiosus Strain Reveals a Highly Dynamic Genome in order to try to extrapolate some features that could ...
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37 views

VDJ sequencing in mice, DNA or RNA?

I am wondering if anyone who is well versed with VDJ sequencing for TCR repertoire analysis (specifically CDR3) would know if DNA or RNA is a better starting material? We are looking at the effects of ...
1
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0answers
21 views

Are the controls for RT-PCR the same as those for RT-qPCR?

I am searching for negative and positive controls for RT-PCR but all the results seem to point towards RT-qPCR. Are the controls the same for both? I have found -RT control No template control ...
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0answers
29 views

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 ...
1
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0answers
14 views

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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0answers
41 views

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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0answers
39 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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0answers
11 views

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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0answers
24 views

SRAP marker sequences

I am looking into SRAP markers and I have realized I am not sure if I need a reverse complement of the sequence given in the article or they are "ready to use" primers?
1
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0answers
1k views

Why do PCR manufacturers recommend assembling the reaction mix on ice?

Many PCR manufacturers recommend preparing the reaction mix for the reaction on ice. For example, here is NEB's recommendation for their Q5 polymerase. Other manufacturers also include similar steps: ...
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0answers
146 views

Using Q solution with ready made MasterMix

I am exploring the possibility of using Q solution (5x) to get rid of non specific bands in PCR. I mostly use a MasterMix and not separate aliquots of dNTPs, Taq, buffer etc. In principle, adding Q ...
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0answers
500 views

What is the meaning of oIMR

In reference to genotyping primers, often some are labeling with 'oIMR'. It seems that this often refers to internal positive control primers, but I'm not sure. What does this stand for?
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0answers
761 views

Interpretation of qPCR results for low expression genes

I am attempting to validate existence of a transcript using 40 cycle qPCR. I designed primers for a unique feature of this transcript, and also designed primers for a sequence in the transcript that ...
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0answers
199 views

Primer heterodimer problem

I design my primers in Primer3PLUS and analyze them in Oligoanalyzer3.1, A big problem that I have this is that I got very nice output in NCBI-Primer BLAST for their specificity but a very negative ΔG ...
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0answers
221 views

rRNA colony PCR amplification not working

We used some universal primers to amplify rDNA in a certain isolated bacterial specie. The problem is that rDNA PCR is not amplifying. Primers were working well in other strains of same bacterial ...
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0answers
277 views

What's a good simple test for cDNA library quality?

How do I assess the quality of a cDNA library? I want to clone CDS copies of genes from a library, but I don't know what's a typical expectation of getting a full length clone even for a shorter ...
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0answers
99 views

Cannot conjugate Biotin-labeled DNA to Streptavidin-labeled solid surface

I have been trying to immobilize DNA by the bioconjugation of biotin and streptavidin, but I cannot get this work. I added EDC and streptavidin to COOH ...
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0answers
62 views

What is the suitable terminology to describe this study approach?

I need to know the correct term(s) which are usually used in the parlance of both biology and bioinformatics for this study approach: About 11 transcripts were investigated using qPCR for a number of ...
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0answers
56 views

condensed protocol for sequencing a portion of human DNA from buccal sample

Anyone have a short & sweet protocol for PCR amplifying a region of human DNA (chromosomal or mt, I don't care) extracted from a buccal sample: including validated primer sequences and preferred ...
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0answers
98 views

DNA fingerprinting

I would like to make my own DNA fingerprint - just for fun to have my "autoportrait":). I was looking around a bit and all the commercial kits you can have are very expensive. Can you suggest me a ...
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0answers
59 views

RT-qPCR of selected differential expressed genes: which dilutions in calibration curve?

I've seen in MIQE guidelines that calibration curves a needed when performing RT-qPCR. I'll use genomic DNA to perform these calibration curves. My question is that I don't know the range of ...
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0answers
2k views

Bacterial 16S rRNA PCR amplification with universal primers

I am doing an experiment to amplify the 16S rRNA gene from bacteria present in gut contents of fish. After extracting DNA, I perform one-step PCR with universal bacterial primers (27F, 1492R) and I ...
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0answers
39 views

The average of Ct values in real-time PCR

The Ct value is the number of cycles when the "PCR amplification curve with the horizontal axis as the number of cycles" and the "predetermined threshold" intersect, and is not ...
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0answers
43 views

3 primers to know if Homozygous, Heterozygous or WT

In this imaginary experiment, I am using T-DNA to make an insertional mutagenesis that will get randomly inserted into the DNA where it might get inserted within a gene, disrupting it. In order to get ...
0
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0answers
55 views

How does plasmid pcr amplifcation work exactly?

Suppose that you have an unwanted gene in a plasmid vector and you want to get rid of it in Gibson assembly. I get the whole concept behind using the primers and essentially making copies that exclude ...
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0answers
15 views

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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0answers
30 views

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 ...
0
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0answers
30 views

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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0answers
76 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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0answers
59 views

Same primers and different melting peaks for two samples (~1°C appart)

In the figure, you can see that melting peaks differ by ~1°C at each sample. Should I be worried?
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0answers
25 views

house keeping gene variation under treatment

I am injecting BCG in mice ears to measure the local inflamation response in the infection side, one ear in the same mouse is kept as control by injecting PBS, in the other one, BCG is injected. When ...
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0answers
28 views

ssDNA library amplification

Assume we are given approx. 80,000 dsDNA fragments of 40 bps length each. I would like to amplify of each of the 80,000 fragments just one particular strand, such that I obtain amplified 80,000 ssDNA ...