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

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13
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2answers
620 views

Are there issues with filling PCR tubes to capacity?

I'm planning to scale up a PCR reaction, and I'm wondering if filling the PCR tubes to the maximum volume of 200 ul would be a problem. It would mean a lot less pipetting as I would only need 1/4 of ...
11
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2answers
8k views

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 ...
11
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1answer
321 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 ...
10
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3answers
3k 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?
10
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2answers
172 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 ...
8
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1answer
1k 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 ...
8
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1answer
121 views

Basic question about multiplex PCR

Let's say I have a DNA sequence with the following structure: $$ 5' - N_n - S_1 - N_{1000} - S_2 - N_{1000} - S_3 - N_n - 3' $$ Here, the $N$s represent stretches of arbitrary sequence of the ...
6
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2answers
213 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 ...
6
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2answers
856 views

Reverse transcription PCR optimization

What is the ideal amount of RNA to use for the RT? and how much cDNA to use then for the PCR? I did RT with a solution of RNA of 0.36 ug/ul. Then for my PCR I used 1 ul of the cDNA obtained and used ...
6
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2answers
5k 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 ...
6
votes
3answers
1k views

Possible reasons for DNA getting stuck in well

I am in the process of Library preparation for Illumina MI-Seq using mtDNA. Using NEB Hotstart LongAmp Polymerase, I was able to obtain upto 10kb amplicons of mtDNA. However, when I switched to a ...
6
votes
1answer
227 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 ...
6
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2answers
162 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 ...
5
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2answers
167 views

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: $\text{Efficiency}^{-(CT\ _{\large\text{interest gene}} - CT ...
5
votes
2answers
159 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 ...
5
votes
1answer
216 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 ...
5
votes
1answer
228 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)?
5
votes
1answer
577 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 ...
5
votes
1answer
32 views

Need to make a library of mammalian coding sequences

I want to clone ~100 different coding sequences from human or mouse into an expression vector to analyze phenotypes in tissue culture. I can do all of the steps, but I'm not sure what I should use as ...
5
votes
1answer
720 views

Equation for accurate prediction of PCR yield

It is a cliche of freshman biology labs to point out that "every cycle of PCR doubles the DNA, so the yield will be $2^{cycles}$ times the template amount". However, if this were true, 1 ng of ...
4
votes
2answers
119 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 ...
4
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2answers
116 views

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 ...
4
votes
2answers
3k 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 ...
4
votes
1answer
129 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 ...
4
votes
1answer
255 views

DNA content in plant 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 a ...
4
votes
1answer
455 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 ...
4
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1answer
1k 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 ...
4
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2answers
557 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 ...
4
votes
1answer
90 views

What are possible reasons for a DNA template not giving amplification?

I am trying to amplify ~10kb length fragments using NEB LongAmp Master Mix. I had been using one particular aliquot of DNA (template amount in each PCR reaction of 10ul being around 60ng) and getting ...
4
votes
1answer
163 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, ...
4
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2answers
108 views

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 ...
4
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2answers
50 views

How to Extract RNA and perform RT-qPCR from very few cells ( ~5,000)?

I am currently conducting an experiment which involves FACS-sorting a specific population of cells using an antibody of interest. In order to validate the type of cells I have collected using this ...
3
votes
2answers
343 views

How are DNA segments selected in PCR?

I understand that in PCR we're able to amplify only selected portions of the DNA... however despite reading it from multiple sources, I cannot figure out how this selection actually takes place. I ...
3
votes
2answers
260 views

Why are some housekeeping genes considered better?

Whenever a PCR is done, we have to use housekeeping genes like GAPDH, to self normalise the values to account for different starting cDNA/DNA. Now there are many different genes like GAPDH, ubiquitin, ...
3
votes
1answer
58 views

How do nicks in the DNA strand affect the success of Long Range PCR?

Long Range PCR using NEB Master Mix - Hot Start Taq was working fine for me (amplicon sizes of ~10kb) but stopped working all of a sudden. Is it possible that many freeze-thaw cycles on the DNA ...
3
votes
1answer
92 views

PCR master mix contents

I am running PCR reactions with different sets of degenerate primers and I want to know what should go into the master mix My usual master mix: DEPC water PCR buffer (-Mg) 25mM MgCl2 10mM ...
3
votes
1answer
133 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 ...
3
votes
1answer
29 views

Quantitative Trait Locus process?

I do not seem to understand the concept of Quantitative Trait Loci(QTL's), can anyone explain it to me in detail? Reading the wikipedia article helped somewhat, but I do not understand it well. What ...
3
votes
1answer
42 views

DNA length and annealing kinetics

I have a mixture plasmids and undesired short linear fragments that share the same sequences. During denaturation and annealing, I would like the plasmids to 'find each other' before annealing to the ...
3
votes
1answer
145 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 ...
3
votes
2answers
529 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?
3
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3answers
607 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 ...
3
votes
1answer
1k 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. ...
3
votes
0answers
30 views

Multiplex PCR, shorter amplicon inhibiting longer amplicon?

I want to run multiplex PCRs for my genotyping, with a primer pair targeting my construct and a primer pair targeting some housekeeping gene (sort of a built-in control). I designed the control ...
2
votes
3answers
91 views

Real-time PCR result interpretation

I performed real-time PCR and 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 ...
2
votes
1answer
53 views

DNA polymerase in PCR (polymerase chain reaction)

Can the DNA polymerase in PCR (polymerase chain reaction) recognize both DNA and RNA for use them as template? I want to know is it possible if my primers bind to an contaminant RNA and then any DNA ...
2
votes
2answers
92 views

Why do we do nested PCR?

Wikipedia: Nested polymerase chain reaction (Nested PCR) is a modification of polymerase chain reaction intended to reduce non-specific binding in products due to the amplification of ...
2
votes
1answer
54 views

Correct/complete representation of RTPCR statistics

In papers reporting a relative quantification of gene expression by RTPCR, I often see a bar chart with mean ± standard error or deviation, with the deviation belonging to biological replicates. This ...
2
votes
1answer
30 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 ...
2
votes
2answers
828 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 ...