Questions tagged [pcr]
Polymerase Chain Reaction is a method of replicating DNA exponentially from even a single molecule.
269
questions
2
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1answer
40 views
qPCR - different results total RNA vs mRNA
I have performed qPCR on a tissue, where I have extracted total RNA and also purified mRNA. I ran the qPCR samples together, and have therefore been exposed to the same conditions except the ...
0
votes
1answer
33 views
How many non-pairing bases can a PCR primer have in directed mutagenesis?
I'm not including a lot of details here, because the problem I'm working with is actually more complex.
Say you have the DNA sequence
5'-...tct gcg gtg gtt ggc att ctg ctg...-3'
Could this sequence ...
1
vote
1answer
43 views
Is there any methods to avoid the pcr control contamination?
Why do I use the primers in the D1-D2 region to amplify the fungus, and using water as a negative control has a weak band that is the same size as the target fragment? When using the ITS primer, using ...
1
vote
1answer
60 views
Transformation and PCR in molecular cloning
After obtaining the recombinant DNA, it is common to transform into E. coli to screen for recombinant DNA and amplify it. But I would like to ask can we amplify it using PCR?
I think there will be 3 ...
1
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0answers
49 views
In what sense is PCR a "nuclear-derived" technique? [closed]
The International Atomic Energy Agency (IAEA) has launched an initiative, ZODIAC, to combat pandemics that originate in animals. In part, this involves the deployment of kits utilising real-time ...
-3
votes
1answer
59 views
Why don't we use hybridization instead of PCR? [closed]
So, I would like to ask, why don't we use just DNA hybridization instead of PCR primer amplification to diagnise some illness?
I know, when you have a really small amount of DNA from virus or cell you ...
0
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0answers
43 views
Lowering annealing temperature in PCR
In the book "Lewin's Genes" there is the following quote:
Lowering the Tm of a PCR reaction—in effect, relaxing the reaction stringency and allowing primers to anneal to not quite
perfect ...
0
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2answers
131 views
How did we do the COVID PCR after its first appear?
I recently learned about the PCR testing and its idea of using primers to replicate the DNA or RNA. By this I assume that we need to know the sequencing of the DNA to make our primers. If that is true;...
2
votes
1answer
76 views
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
76 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
votes
1answer
54 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
45 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 ...
1
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0answers
50 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 ...
1
vote
1answer
130 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
vote
2answers
55 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
83 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
votes
1answer
61 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
votes
1answer
91 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
vote
1answer
120 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
votes
1answer
127 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
vote
1answer
138 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
218 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
votes
2answers
96 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 ...
9
votes
2answers
242 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
votes
1answer
74 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'-...
1
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0answers
40 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
votes
1answer
54 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
votes
1answer
44 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?
1
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2answers
363 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
votes
1answer
75 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.
1
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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
votes
1answer
97 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
votes
0answers
40 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
45 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
votes
2answers
118 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
16 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
78 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
87 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
votes
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
votes
1answer
64 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
votes
2answers
74 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
vote
1answer
50 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
votes
1answer
64 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 ...
1
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0answers
30 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 ...
7
votes
2answers
162 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
105 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
votes
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
votes
1answer
60 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
votes
1answer
94 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 ...