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

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2answers
43 views

Primers for human tissue? [on hold]

If I wanted to examine human tissue samples, such as hair, blood, fingernails, etc, for their DNA, what primers would I use for the amplification of the DNA when extracted(PCR)? edit: To look at the ...
1
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1answer
40 views

Is the amount of dNTPs rate limiting for very long PCR products?

I'm using the Q5 system and I'm PCRing a product that will have a final length of around 9500 bases. I have noticed that the product is there but very faint. The primer seems to be used up. So my ...
1
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1answer
29 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 ...
-3
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0answers
21 views

What are the rules for primer design [on hold]

I am looking for a (most complete, not to say ultimate) set of rules that one should follow to achieve the best performing PCR primers (aside from being compatible to the desired target sequence). ...
1
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1answer
15 views

18S rRNA sequence

I have this phenomenon that the human 18S rRNA reverse transcribed with polydT oligos serve as faithful RTPCR normalizers, tested with better known house keepers. I want to know what I'm amplifying ...
1
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0answers
10 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 ...
0
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0answers
9 views

What is the Magnitude of Nonspecific Oligo Binding at Low Temperatures

I am working on designing a protocol to capture genomic sequencing bound oligos (the part in question is identical to primer binding of genomic DNA during a PCR reaction). I'm wondering if anyone can ...
3
votes
2answers
212 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
0answers
24 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 ...
3
votes
1answer
23 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 ...
1
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0answers
26 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 ...
3
votes
1answer
29 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 ...
2
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0answers
13 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
votes
1answer
45 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 ...
1
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0answers
41 views

Closing a Plasmid by Ligation

I have been having trouble with what should be a fairly simple ligation. I had a plasmid that I needed to cut part out of, so I designed a couple PCR primers to do that. The plasmid already had a ...
0
votes
0answers
15 views

Solid mouse (C57BL/6) WT primers

I am trying to get a good pair (or better yet, library of pairs) of primers that give me one band in WT mice (C57BL/6). Do you know where I could find such a thing? Alternatively, I thought of just ...
2
votes
1answer
49 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 ...
1
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0answers
42 views

Primer Designing [closed]

I have a basic idea about the requirements in primer designing. Basically my knowledge is limited to theoretical knowledge and have no experience in actual primer designing. And also I have a basic ...
2
votes
0answers
21 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 ...
1
vote
2answers
37 views

How does one calculate how to dilute a solution to working strength? [closed]

If I'm loading a 3.5ul PCR onto an agarose gel, how do I calculate how much of the 6x loading dye to add?
1
vote
2answers
49 views

DNA barcoding and real-time PCR

I recently read an article on how DNA barcoding was used to identify species present in health products. I also read an article about how Real-Time PCR was used to identify meat species in meat ...
0
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0answers
25 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 ...
1
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2answers
32 views

Can iGEM distribution parts be directly PCR'd?

The iGEM DNA Kit Plate Instructions say that there is only 2-3ng of DNA per well, which is already miniprepped and in plasmids. Then it says that "there is not enough DNA in each well to perform ...
0
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0answers
25 views

Real Time PCR Test Chemistry

I am working on building a real time PCR machine. Is there any chemistry set that I can purchase online to: Identify if PCR amplification was successfully done. Test fluorescence dyes that could ...
2
votes
1answer
28 views

Concentration of degenerate primers should you dilute to?

I'm a little embarrassed to ask but when you have for example four degenerate primers and the end protocol says that the final primer concentration should be 10 µM working stock, should you make the ...
0
votes
1answer
31 views

PCR cycle problem [closed]

IF I began a PCR cycle with 5 copies of a particular DNA section, and copied the section by PCR, for 6 cycles, how many copies of the DNA (include the originals) would I have by the end of these ...
0
votes
1answer
52 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). ...
8
votes
1answer
115 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 ...
2
votes
1answer
112 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 ...
5
votes
1answer
31 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 ...
2
votes
1answer
202 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 ...
3
votes
1answer
42 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 ...
4
votes
1answer
80 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 ...
3
votes
2answers
332 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 ...
6
votes
3answers
768 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 ...
1
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0answers
44 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 ...
5
votes
1answer
579 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 ...
13
votes
2answers
535 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 ...
1
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0answers
39 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 ...
1
vote
1answer
115 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
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1answer
35 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 ...
3
votes
1answer
73 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 ...
1
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0answers
23 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 ...
5
votes
2answers
135 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 ...
3
votes
1answer
116 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 ...
1
vote
2answers
390 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 ...
2
votes
3answers
79 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 ...
1
vote
1answer
147 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 ...
4
votes
2answers
2k 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 ...
2
votes
1answer
54 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 ...