Questions tagged [pcr]

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

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14
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2answers
2k 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 ...
14
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2answers
18k 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 ...
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4answers
21k 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 ...
11
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1answer
406 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
19k 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
261 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 ...
9
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3answers
1k views

Is Melanin a PCR Inhibitor?

I have been growing B16F10 Mouse Melanoma cells. I need to extract the genomic DNA and do PCR to amplify a specific region. However, no matter what temperature or magnesium concentration I use, I have ...
8
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1answer
2k 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
311 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 ...
8
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2answers
720 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 (...
7
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2answers
2k views

Good pipetting technique?

Good pipetting technique is essential for many biologists, but it can be hard to get right. When I take 1 µl of liquid using a micropipette, I seem to always take less than 1 µl, and that amount is ...
7
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2answers
1k 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 ...
7
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2answers
17k 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 ...
7
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1answer
342 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 ...
7
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1answer
1k 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 homo-...
6
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2answers
337 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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3answers
9k 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
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2answers
604 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 _{\large\text{...
6
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2answers
1k 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 ...
6
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1answer
318 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
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2answers
247 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 ...
5
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3answers
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 ...
5
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3answers
27k 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 ...
5
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2answers
247 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
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1answer
6k 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 ...
5
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1answer
450 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
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2answers
2k 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 ...
5
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1answer
31 views

Is there a biological or technical reason for a 40 Ct cutoff in nucleic acid detection assay?

I have developed and validated a modified a nucleic acid test (NAT) for SARS-COV-2 detection using real-time RT-PCR (aka rRT-PCR, aka RT-qPCR). My assay is not for diagnostic use, but for donor ...
5
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1answer
38 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 ...
4
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2answers
175 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
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1answer
216 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
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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 ...
4
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1answer
4k 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
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1answer
641 views

Freezing after restriction digest

Brief question: I have performed a double digest with heat inactivated restriction enzymes with no star activity. I usually purify the DNA (PCR inserts and linearised vector) using a kit or gel ...
4
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2answers
2k 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 ...
4
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1answer
1k 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
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1answer
390 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, 10&...
4
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3answers
253 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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1answer
118 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 ...
4
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2answers
2k 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?
4
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3answers
2k 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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0answers
75 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 ...
3
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2answers
60 views

RT-PCR: Not seeing how it can measure mRNA expression levels

From my understanding, in RT-PCR we start off with an mRNA molecule, use the enzyme reverse transcriptase to create a cDNA copy (hence creating a double-stranded mRNA / cDNA hybrid), degrade the mRNA, ...
3
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1answer
113 views

Why do we need to amplify DNA sequences?

I am learning about biotechnology. I have no education in biology or chemistry and am simply interested in the subject of biotechnology. I am wondering why we need to have multiple copies of a piece ...
3
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1answer
175 views

Can primers for PCR be duplicated?

Complete beginner question here, don't laugh: If I have some primers that have been synthesized, and I am close to running out of them, is there any way to duplicate them / amplify them / synthesize ...
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2answers
2k views

In Sanger sequencing, why do we resort to cloning? Why doesn't PCR suffice?

I understand that in Sanger sequencing we can clone our fragments with the help of e.g. bacteria to make multiple copies of our fragments for further analysis. I also understand cloning can be a ...
3
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1answer
129 views

Walk me through microsatellite markers and PCR

Three polymorph microsatellite markers are used to try and narrow down the location of a disease locus, with the use of PCR with 2 flanks on each side of the actual polymorphic area. The PCR product ...
3
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2answers
690 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
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1answer
315 views

Why is the Tm defined as the temperature at which 50% of dsDNA has changed into ssDNA?

In molecular biology, Tm is defined as the temperature at which 50% of dsDNA is converted to its single stranded form. Intuitively it would seem that the melting temperature should have been defined ...
3
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2answers
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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