Questions tagged [pcr]
Polymerase Chain Reaction is a method of replicating DNA exponentially from even a single molecule.
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1answer
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Using PCR to add the overhangs to gBlocks for NEB HiFi
Does adding the overhangs to gBlocks for NEB Hifi using PCR pose a big problem? The IDT website suggests that one should design the gBlocks with the overhangs. However, if I did not do this, will it ...
0
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0answers
39 views
The average of Ct values in real-time PCR
The Ct value is the number of cycles when the "PCR amplification curve with the horizontal axis as the number of cycles" and the "predetermined threshold" intersect, and is not ...
3
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1answer
51 views
Polymerase Cycle Assembly (PCA) is not working (Only primrs in EF Gel)
Helllo All 👋!
I am working trying to assemble dsDNA fragments (for further golden gate domestication) via polymerase cycle assembly (PCA). After designing the required oligonucleotides with overlap ...
4
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0answers
43 views
Unknown Issue with qPCR Standard
I am currently doing the ground work for a SELEX experiment and I am attempting to verify my standards for the starting library. I ran a reaction with samples containing 50, 5, 0.5, and 0.05ng of my ...
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0answers
49 views
What are the causes of gene amplification in archaea?
I was studying this article about Genome Sequencing of a Genetically Tractable Pyrococcus furiosus Strain Reveals a Highly Dynamic Genome in order to try to extrapolate some features that could ...
2
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1answer
110 views
Detection of primer dimer on RT-PCR?
I usually do RT-PCR test for covid-19. Curves for Patients and Positive CTRL mostly appeared at 20 to 25 Ct. Sometimes, there are some curves that emerge at the last 5 cycles. Some argue that they are ...
1
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2answers
52 views
Why there is Cp/Ct (crossing point) value for negative sample in COVID-19 RT-PCR?
I am from IT/ Engineering background and have some confusion in RT-PCR. So far my understanding we extract RNA from test specimens and transcribed it into complementary DNA (cDNA), and inside PCR DNA ...
6
votes
2answers
77 views
Which method of gene amplification for toehold switches?
My team and I are from a high school and are planning to carry out some research investigating some toehold switch riboregulators which we have designed in silico. However, we have little experience ...
2
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1answer
52 views
Is it possible to use two forward primer and one reverse primer?
I have eGFP and the gene fragment i assembled by pcr.The next step is that i have to join my fragment fragment into 3'p end of the eGFP.I have three primers FP1 is forward primer for eGFP, FP2 has ...
3
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1answer
88 views
Is it possible to use PCR to test for Machado-Joseph disease?
Machado–Joseph disease (MJD) is a rare inherited neuromuscular disease that is caused by a mutation in the gene ATXN3. The protein encoded by this gene contains "CAG" repeats in the coding ...
1
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1answer
73 views
Does the false positive covid-19 PCR % referred to by Surkova & Nikolayevskyy in The Lancet mean % of all tests, or % of positive tests?
In False-positive COVID-19 results: hidden problems and costs it is said:
The current rate of operational false-positive swab tests in the UK is
unknown; preliminary estimates show it could be ...
2
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1answer
88 views
SARS-COV-2 replication speed
What is the speed of replication of SC2? Any information, including in vitro data would be appreciated.
I would be interested to know the length of SC2 eclipse period, latent period and something like ...
1
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1answer
99 views
Nomenclature of substrates for DNA synthesis
I have read in my school textbooks that both deoxyribonucleoside triphosphate and deoxynucleotide triphosphate are used in DNA Replication as substrates.
However, it is unclear to me whether the terms ...
4
votes
2answers
117 views
How does PCR mutagenesis add restriction site near the gene of interest?
I have been learning about PCR mutagenesis to add restriction site right next to the gene of interest using a primer that's attached with single stranded restriction site (first image). I have drawn ...
2
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2answers
92 views
How to assemble three 60mer nts by pcr?
Good morning,
I am new to molecular biology. The question might be silly but i would like to know the answer. I have three 60mer single strand synthetic oligonucleotide. Namely Tag 1 - 3. My goal is ...
8
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2answers
172 views
Do the primers used for Covid-19 test work with all new strains?
We keep reading news of new Sars-CoV-2 strains, some of them allegedly able to evade vaccine-induced Igs. That's not unexpected, specially for a single-stranded RNA virus. However, it seems to me that ...
0
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1answer
64 views
Gibson assembley for small fragments?
I am new to the Gibson assembly, I know how I need my plasmid but main problem is I don't have short tags and linker so I wanted to do Gibson assembly?
5'-...
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0answers
37 views
VDJ sequencing in mice, DNA or RNA?
I am wondering if anyone who is well versed with VDJ sequencing for TCR repertoire analysis (specifically CDR3) would know if DNA or RNA is a better starting material? We are looking at the effects of ...
0
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0answers
43 views
3 primers to know if Homozygous, Heterozygous or WT
In this imaginary experiment, I am using T-DNA to make an insertional mutagenesis that will get randomly inserted into the DNA where it might get inserted within a gene, disrupting it.
In order to get ...
0
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1answer
50 views
PCR and large-scale PCR
I have performed PCR with 50 μL, which showed a clear band in the gel electrophoresis. I then performed large-scale PCR with 3650 μL, which showed an unclear band in the gel electrophoresis. What can ...
0
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1answer
41 views
Can Taq polymerase be stored with PCR primers?
I don't think it's possible for TAQ to be stored with the primers, but I'm not sure. This storage wouldn't be long-term (a few days, or a week at most). Thoughts?
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2answers
204 views
What is an “open system” when it comes to PCR machines?
I have heard the term "open systems", such as here when it comes to RT-PCR machines, but I'm not 100% sure what this means, does this mean machines you can purchase yourself? Such as this ...
0
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1answer
47 views
What is the significance of running an uncut plasmid on electrophoresis gel?
What is the significance of running an uncut plasmid on electrophoresis gel?
In this case we are talking about inserting a gene into plasmid, which then goes under PCR and then electrophoresis.
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0answers
57 views
How does plasmid pcr amplifcation work exactly?
Suppose that you have an unwanted gene in a plasmid vector and you want to get rid of it in Gibson assembly. I get the whole concept behind using the primers and essentially making copies that exclude ...
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0answers
21 views
Are the controls for RT-PCR the same as those for RT-qPCR?
I am searching for negative and positive controls for RT-PCR but all the results seem to point towards RT-qPCR. Are the controls the same for both?
I have found
-RT control
No template control
...
0
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1answer
92 views
Does “RT-PCR” denote “real-time PCR” or “reverse transcription PCR”?
I often find the term "RT-PCR" used in articles without any further qualification. Searching for the meaning of the acronym sometimes leads to "real-time polymerase chain reaction" ...
3
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0answers
39 views
Why is my DNA band bulging?
This is the only image my the TA was able to get for us. And, we're using it for our lab report. The image isn't even ours. It's another group's image that we're sharing. But I don't understand why in ...
5
votes
1answer
33 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 ...
0
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2answers
62 views
Polymerase Chain Reaction Specifics
While going over PCR in my biology lecture this week I have come across a few questions I have about this process.
First, since PCR focuses on trying to replicate a specific targeted DNA sequence many ...
0
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0answers
15 views
LAMP (isothermal amplification), hydroxy naphtol blue , disodium salt" or just hydroxy naphtol blue?
I am reproducing this work on LAMP (isothermal amplification)
Article A new visually improved and sensitive loop mediated isotherm...
,
but I am a bit confused about the difference between "...
-1
votes
2answers
67 views
Why PCR primers don't amplify beyond the target region? [closed]
I have been doing PCR for a while. I spared a bit of time to look it up but could not find an answer. So, for sanger sequencing, if we have a primer, it can amplify about 800-1000 bp. But, if you ...
3
votes
2answers
77 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, ...
0
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2answers
66 views
Meaning of “standard reactions” in a DNA extraction procedure description
From a DNA extraction procedure description (an in-house pharma document I'm translating into Russian):
Preparation of Standards
All the standard reactions should be prepared at least in duplicates. ...
0
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1answer
57 views
Meaning of “optical” in “Optical cap, 8X Strip” (used in qPCR)
I'm translating an English document that lists equipment used in a qPCR procedure:
Reagents/Materials
Optical cap, 8X Strip
I googled and found that the meaning of this line is "a strip of 8 ...
0
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2answers
52 views
High concentration of primer in PCR
Why is it that higher concentration of primers than the DNA template in PCR will favor the annealing of a template to a primer?
1
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1answer
44 views
Humic Acid and PCR
Quite a few papers claim humic acid is an inhibitor of PCR reactions. I understand this is true when working with soil microbes, but how does it qualify to be a PCR inhibitor in general (i.e when not ...
0
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1answer
53 views
Are specific primers or detectors, or both, used in COVID-19 tests?
I am trying to learn about the rRT-PCR testing procedure used to test for COVID-19, but I am slightly confused on one point. Are highly specific primers used with a non-specific detector, or are ...
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0answers
29 views
Is there a difference between a consensus PCR and a pan PCR?
I know that a multiplex PCR uses multiple sets of primers in one reaction to amplify multiple targets.
But what is a PCR called that uses generic primers to amplify multiple targets? Is this a ...
5
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0answers
88 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
votes
2answers
96 views
What are the trade-offs between polymerase chain reaction (PCR) vs Loop-mediated isothermal amplification (LAMP)?
I've heard that LAMP could be a good alternative to PCR for DNA strand amplification and detection because the equipment required is cheaper, more portable, and the amplification is faster since all ...
0
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1answer
28 views
Swing bucket for PCR
I see a swing bucket rotor for PCR trips and don't know what step is using? What difference if I use fixed - angle? In addition, Which step I can use swing bucket instead fix - angle in molecular ...
0
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1answer
57 views
How to calculate the quantification of qPCR to the equivalent of the number of nuclei in fungi?
I have a question about the quantification through qPCR. The my question is: if I made the qPCR of a fungal functional gene, how is possible to obtain from the quantification number (in nanogram) the ...
0
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1answer
87 views
How to design the primer when you don't have GC?
I have treated my RNA with sodium bisulfite so all my cytosine is converted into thymine. Then I used that RNA for my RT reaction. I have a cDNA template without Cs. Now I am trying to amplify that ...
1
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1answer
41 views
PCR markers for C57B6
Do you know any primers that can be used to genotype mice and check if they are still C57B6? I'm concerned about genetic drift in my colony. I bought a breeding pair 3 years ago and expanded. I wanna ...
0
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1answer
22 views
Minimum amount of RNA for cDNA conversion and qPCR
I searched several protocols for reverse transcription of RNA and all suggest using RNA concentrations of 1ug. Could a lower amount result in inferior transcript representation and therefore decreased ...
2
votes
1answer
74 views
Taq Polymerase's stability at high temperature
I was asked a question as to what is so special with Taq Polymerase that makes it quite stable at high temperatures though its functioning is the same as other DNA polymerases like that of mesophiles. ...
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1answer
35 views
Is chromosomal DNA more likely to interfere with restriction mapping or PCR analysis in E. coli?
We are characterizing YADH-1 in my biochemistry lab course. Over the course of the first weeks, we harvested E. coli cells, and isolated plasmid DNA via alkaline cell lysis. A previous exam for the ...
0
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1answer
70 views
What would be the effect of excess taq polymerase on the PCR?
I just had one question regarding the possible effect of putting to much Taq polymerase in my PCR tube?
Instead of 5µl I put 50µl (10x more).
Do you think it will have a bad effect on the reaction??...
3
votes
1answer
135 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
votes
1answer
180 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 ...
