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

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Derivative form of HRM results

I used in the laboratory the Rotor Gene 6000 for a real time PCR and HRM analysis. When i analyzed the results of melting step, the derivative plot in the y-axis used the dF/dT . On the other hand ...
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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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244 views

How to calculate virus titre from qPCR

I harvested some lentivirus from 293T cells and want to titre the result. I infected 293T cells on a well plate with 400,000 cells per well which I infected with virus stock, and 1 in 10, 100 and 1000 ...
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Real time PCR normalization algorithm

When performing normalization of real time PCR results, I found two ways of doing it: In my lab they follow the next layout: $efficiency^{-(CT\ interest\ gene - CT\ housekeeping)}$ each time ...
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38 views

gene expression fold change threshold limit

When interpreting qRT-PCR results, which results are acceptable as fold changes, which are acceptable for housekeeping genes? e.j. a values of 0.8 raw fold changes for a "housekeeping" gene is ...
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81 views

Real time PCR standard curve

As blunt as possible: when performing real time PCR it is a routine step to run one PCR in order to plot a "standard curve" with several decreasing dilution ratios from your sample. what is the real ...
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25 views

Real time PCR result interpretation

i performed a real time PCR. I was looking for expression fold changes for 2 genes and i had two sample pools, one treated and the other not treated (for each gene). The problem is that my ...
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1answer
38 views

Real time PCR parameter CT

When puting a real time PCR, parameter CT, which means threshold cycle, is used. What does it mean really? according to wikipedia "The number of cycles at which the fluorescence exceeds the threshold ...
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40 views

PCR enzyme units or concentration?

When performing PCRs, usually in every protocol enzyme amount is specified as 1.25 U as optimal per 50 uL reaction. Then, when running 25 uL PCR reaction should i use 1/2 he amount the enzyme or ...
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60 views

No bands show up in gel electrophoresis, not even marker

I haven't seen any gel lanes in neither my PCR samples nor the marker itself when photographing them. This is not my first time performing 1.2% agarose gel electrophoresis. I see gel lanes when I ...
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31 views

parallels or replicates?

I am writing an article where I describe real-time PCR experiments. My collaborator, after reviewing the article, consistently replaced the word "parallels" with "replicates". Are they not synonymous? ...
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Is there a strong reason to be sceptical about the “cured HIV patient” being reported by mainstream media?

There's a story going round the news about a baby that was, apparently, cured of HIV using a cocktail of drugs at an early age. The story piqued my interest, but details seem scarce. One of the main ...
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214 views

Why is the number of PCR cycles limited?

I've been told that the maximum number of cycles in PCR is between 20 and 30. Is this true, and what are the reasons for this limitation?
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47 views

How does sequence independent single primer amplification work?

I'm having difficulty finding an explanation of how single primer amplification of DNA works in the literature available to me, can anyone explain the methodology and what it accomplishes?
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179 views

Effect of single nucleotide deletion or insertion on primer annealing

How is primer annealing, and, consequently, PCR amplification affected by single nucleotide deletion or insertion inside the primer ? Imagine a primer like this: GCGTCATAAAGGGGACGTG (primer) and ...
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76 views

Real-time PCR delay in Cq due to insertion SNP in primer

I am collecting evidence, even anecdotal, how does single nucleotide deletion or insertion in primer region affect the outcome of real-time PCR. I am most interested in how much there is a delay in ...
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41 views

DNA content in seeds vs. fruit flesh

Is there a publication comparing DNA yield and / or PCR-amplifiability after extraction from fruit flesh (like apples, oranges, cherries etc) in comparison to seeds of the same fruits ? I would prefer ...
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43 views

SNP genotyping using PCR

I read this wikipedia article on SNP genotyping and wasn't able to understand this part : In examining the results, if a genomic sample is homozygous, then the PCR products that result will be ...
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230 views

Deletion errors with Phusion Polymerase?

I was wondering if anyone has ever seen large deletions (~40-50 bp) occur when using phusion polymerase to clone a gene. I have recently tried using Circular Polymerase Extension cloning to clone a ...
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36 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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1answer
55 views

pfu Turbo DNA polymerase AD

What is the duration (Life") of pfu turbo DNA polymerase AD. I am trying o Amplification a 3.3kb fragment and the PCR schedule is 6 hours long. The manufacturers notes just specify that its activity ...
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1answer
26 views

Difference vector target and genomic target

In Pfu turbo DNA Polymerase AD information sheet (and possibly in many other) different amplification targets are divided in Genomic DNA targets, Vector DNA targets and cDNAs. What is the difference ...
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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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465 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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DNA cloning with gateway primers

I tried to clone a 5 kb gene with few success. The primers used are this OLIGO start len tm gc% any 3' seq LEFT PRIMER 1 22 62.53 50.00 4.00 2.00 ...
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568 views

Basic step by step methods for PCR & Gel electrophoresis class

I'm teaching a class how to do PCR & gel electrophoresis soon and would like it if you could check through my basic step by step instructions - it's a while since I've done one. Is there anything ...
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27 views

Linearity with SuperScript® VILO™ Master Mix?

I would like to know your experiences with SuperScript® VILO™ Master Mix. According to de manufacturer, this kit maintains linearity from 1 pg to 2.5 µg in a 20 µL reaction. enter link description ...
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227 views

Does bleach destroy RNAse activity, and if so, how does it do it?

I am working with RNA samples, and I'm trying to be very careful about RNAse contamination. I have some questions about bleach, though. Some people say that a solution of bleach is enough to destroy ...
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757 views

When designing primers how important is the GC clamp?

I'm designing a set of primers and reading about the principles of primer design one of which is: GC Clamp: The presence of G or C bases within the last five bases from the 3' end of primers (GC ...
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526 views

What is the purpose of Y-shaped adapters in Illumina sequencing?

Y adapters different sequences to be annealed to the 5' and 3' ends of each molecule in a library. The arms of the Y are unique, and the middle part, connected to the DNA fragment, is complementary. ...
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47 views

Creating expression template from PCR: What annealing temperature to use?

I am creating a transcription template for expression in PURExpress and was confused about the annealing temperature to use. I have two primers with the T7 promoter and an RBS on the forward primer ...
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61 views

qrt-pcr and short fragments

I plan to measure the effects of Tenofovir a Nucleotide analog reverse-transcriptase inhibitors useing qrt-pcr with HIV-RT as the rt enzyme. Tenofovir causes early termination of the reverse ...
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30 views

Amount of reverse transcriptase in µg or mM for qRT-PCR

I am trying to calculate a titration amount for a molecule which I would like to use in my PCR samples. Different molecules have different densities so I would like to calculate the appropriate ...
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1answer
26 views

qTERT-pcr like qRT-pcr

Is it possible to use a biologically active Telemorease Elongation Reverse Transcriptase (TERT) in the place of the Reverse Transcriptase (RT) for quantitative Reverse Transcriptase pcr (qRT-pcr)? Yes ...
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442 views

How prevalent is Taq polymerase in adding 3' A overhangs to the PCR product?

I am conducting a mutagenesis on a gene in vivo of which I need to ligate into an expression vector. The primers I have designed overlap restriction sites of which I plan to use to ligate into the ...
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91 views

RACE pcr product

I'm a molecular bio novice, and I'm trying to understand the differences between regular PCR and RACE PCR. If it wanted to sequence my PCR product that might be hundreds, if not thousands of bps ...
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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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1answer
107 views

Effect of doubling volumes of PCR reagents

We ran PCR with a positive control and two lanes of sample. We used the same sample, but in one PCR mixture we used a total volume of 50μL (25μL Taq, 5μL forward&reverse primer each, ...
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1answer
144 views

Can Taq polymerase be used instead of polymerase Vent exo (-)?

Instead of using polymerase Vent exo (-), can I go with the usual Taq polymerase? Do the PCR conditions change (the temperature and master mix concentrations ) in the these two conditions? Do the ...
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Low temperature PCR

We're trying to do emulsion PCR using HA-coated polystyrene beads and we're noticing that the beads are seeing drastic issues with thermal degradation above 90C. As PCR has an unfortunate requirement ...
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138 views

Analysing the results of real-time PCR

I want to evaluate the level of gene expression by real-time PCR. I have five controls that are "clinically" the same. I calculated the "fold change" of the target gene regarding each control. What ...
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624 views

How to clone and sequence a gene transcript of unknown sequence?

How might I go about amplifying a gene transcript (mRNA) from animal tissue of which little is known about the genome? In some applications, I have used reverse transcriptase PCR to amplify all mRNA ...
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1answer
192 views

How are DNA polymerase error rates measured?

It is well known that the first DNA polymerase, Taq, is quite error prone. Newer generation commercial enzymes that have either been isolated from different thermophile species or have been improved ...
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163 views

Is there a detectable amount of bacterial DNA in the blood of infected persons?

With which bacterial infection in humans has it been shown that bacterial DNA can be found in the blood? If any is found it is likely not to be very much, and even difficult to distinguish from ...
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1answer
350 views

Primer Dimer / Hairpin Algorithms

What are the algorithms / methods in use for the calculation of primer dimers and hairpins? As an example, IDT's OligoAnalyzer tool will generate these analysis given particular sequences. The ...
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106 views

Sequencing from PCR

As far as I understand it, PCR can be used to make many copies of one gene. My question is, is it possible to sequence DNA after PCR and is it easier than sequencing it via other methods. If it is ...
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140 views

PCR primer in highly repetitive region

Is there any way to design a PCR primer if region of interest is highly repetitive (tens of kilobases only SINE's & LINE's)?
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188 views

PCR amplification and error propagation

Say I have one dsDNA that undergoes normal PCR (where amplification is exponential). If there is a mistake, say a G is swapped for an A during the 2nd round of replication, what percentage of the ...
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141 views

What is the least costly method to generate sequential amino acid deletions?

I'm looking to generation sequential deletions from a gene of interest. The total size of this region is 8 amino acids. I'm trying to determine which portion of this region is necessary within the ...
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Does an annealing temp higher than primer's Tm contribute to primer dimer?

I am attempting to reproduce results from a number of journal articles all referring to the same SNP. In doing this I'm using the same primer set outlined in the articles. When I attempted a run the ...