Questions tagged [pcr]
Polymerase Chain Reaction is a method of replicating DNA exponentially from even a single molecule.
244
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0answers
744 views
Interpretation of qPCR results for low expression genes
I am attempting to validate existence of a transcript using 40 cycle qPCR. I designed primers for a unique feature of this transcript, and also designed primers for a sequence in the transcript that ...
1
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2answers
3k views
What happens when we re-start a PCR reaction?
Recently when my PCR reaction was running there was power fluctuation and the entire lab was blacked out for a few minutes and unfortunately PCR that I was running got switched off. So, would it be ...
1
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1answer
441 views
Design arbitrary degenerate primers (with non-binding criteria)
I would like to design a number of arbitrary degenerate primers (primers with variable bases, e.g. NGATWGCTSATNGC) for a TAIL-PCR.
I would like to be able to ...
0
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1answer
1k views
Which pair of primers should be used to amplify the ORF in PCR? [closed]
So I want to choose the correct set of pair of primers to amplify the ORF of the gene that corresponds to amino acids in a protein. The start and stop codons are underlined. (I know that these need to ...
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 ...
1
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0answers
193 views
Primer heterodimer problem
I design my primers in Primer3PLUS and analyze them in Oligoanalyzer3.1, A big problem that I have this is that I got very nice output in NCBI-Primer BLAST for their specificity but a very negative ΔG ...
1
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1answer
762 views
DNA extraction methods for hair?
TLDR: Can anyone state a extraction and isolation method(s) for genomic DNA for hair that will be used for PCR, in detail is preferable since I am a novice.
I tried googling for DNA extraction ...
-1
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2answers
203 views
Primers for human tissue? [closed]
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 ...
3
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1answer
1k 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 ...
2
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2answers
5k 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 ...
1
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1answer
46 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 ...
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0answers
221 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 ...
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, ...
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 ...
3
votes
1answer
167 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 ...
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0answers
277 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
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1answer
411 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
17 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
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1answer
359 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 ...
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0answers
174 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
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1answer
90 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
149 views
DNA polymerase in PCR (polymerase chain reaction)
Can the DNA polymerase in PCR (polymerase chain reaction) recognize both DNA and RNA for use as a template?
I want to know is it possible if my primers bind to contaminant RNA and then any DNA ...
2
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0answers
60 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 ...
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2answers
14k 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?
2
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2answers
402 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 ...
1
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0answers
99 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
87 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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1answer
270 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 be ...
2
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1answer
736 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
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1answer
177 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 ...
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 ...
2
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1answer
995 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
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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 ...
2
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1answer
2k 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
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1answer
343 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
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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 ...
2
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2answers
857 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
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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 ...
2
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0answers
101 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 ...
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 ...
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 ...
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0answers
62 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
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1answer
410 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
58 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
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1answer
547 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 dNTPmix
...
1
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0answers
56 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 ...
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{...
3
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1answer
299 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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2answers
1k 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
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3answers
516 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 ...
