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
412 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
votes
2answers
6k 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
votes
1answer
282 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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2answers
156 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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2answers
1k 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?
8
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1answer
840 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 ...
7
votes
3answers
376 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
2answers
206 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
votes
2answers
759 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
3k 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
1answer
194 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 ...
5
votes
2answers
152 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
209 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
196 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
446 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
30 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
231 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
112 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
votes
2answers
109 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
1k 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
96 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
343 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
votes
1answer
893 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
votes
2answers
372 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
54 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
122 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
96 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 ...
3
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2answers
300 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
1answer
57 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 ...
3
votes
1answer
31 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
52 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
94 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
97 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 ...
3
votes
1answer
114 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
236 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
votes
3answers
425 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
967 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
84 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 ...
2
votes
2answers
94 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: $efficiency^{-(CT\ interest\ gene - CT\ housekeeping)}$ each time ...
2
votes
1answer
28 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
641 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 ...
2
votes
1answer
53 views

(Updated question) Problem with Fold-change mean compared to absolute data (in qPCR)

For the problem below I am aware of the statistics involved but just can't get my finger around the following: In biology we use qPCR to measure gene expression or basically number of mRNA copies. It ...
2
votes
1answer
83 views

Effect of 260/230 values on PCR

To what extent does the 260/230 value affect the efficiency of PCR reactions ? Do the constituents leading to a low 260/230 value (EDTA, guanidine salts and oligosaccharides) inhibit PCR ? I have been ...
2
votes
1answer
195 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 ...
2
votes
1answer
51 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 ...
2
votes
0answers
25 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 ...
1
vote
1answer
91 views

A question related to qPCR analysis

Here is the thing. I am using a method, called TU-Tagging, to isolate cell type specific RNA. You can find more about the method here: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2783170/ To explain ...
1
vote
2answers
229 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 ...
1
vote
1answer
29 views

PCR directly on DNA binds to Ampure XP

During the library preparation it's possible to conserved Ampure beads during the different enzymatic reaction (it's the with beads strategy for improving yield, Fisher et al. Genome Biology 2011). ...
1
vote
1answer
28 views

Standard curve for real time

When performing a standard curve for some new primers to test for fold change, is it necessary to run the standard curve? I mean, in case your experiment has several genotypes for the same ecotype ...