Questions tagged [pcr]
Polymerase Chain Reaction is a method of replicating DNA exponentially from even a single molecule.
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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 ...
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where can i introduce my insert in the plasmid?
I assembled three different DNA fragments together. I want to do the gibson assembly to introduce my fragments into the plasmid. Main point is i don't want to use the restriction sites. I am using ...
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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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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 ...
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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 ...
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1answer
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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 ...
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1answer
36 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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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 ...
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1answer
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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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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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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
...
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1answer
87 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" ...
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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 ...
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1answer
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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 ...
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2answers
45 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 ...
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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 "...
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2answers
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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 ...
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2answers
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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, ...
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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. ...
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1answer
51 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 ...
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Construct aptamer library with exact number of members
I am asked to construct an aptamer library of exactly 1024 members, each with a unique V.
I have two questions regarding this; firstly, is it wrong to construct an aptamer library using a column with ...
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2answers
46 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?
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42 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 ...
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1answer
47 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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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 ...
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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 ...
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2answers
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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 ...
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How to recalibrate qpCR reading when incorrect qty of solution is added during DNA elution step?
I have 20 viral DNA samples collected for 6 time points. The DNA content varies over time My DNA extraction protocol step suggests that in the last step of the protocol during elution I had 100uL of ...
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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 ...
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1answer
56 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 ...
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1answer
85 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 ...
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1answer
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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 ...
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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 ...
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1answer
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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
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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 ...
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1answer
51 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??...
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1answer
118 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 ...
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1answer
175 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 ...
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1answer
317 views
Why is the Tm defined as the temperature at which 50% of dsDNA has changed into ssDNA?
In molecular biology, Tm is defined as the temperature at which 50% of dsDNA is converted to its single stranded form. Intuitively it would seem that the melting temperature should have been defined ...
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What does the final (Illumina) RNA-seq library contain? ss cdNA or ds cDNA?
I guess the oligos on Illumina's flow cell only hybridize to to single-stranded cDNAs (sscDNA).
However, when I read the TruSeq protocol (googled: TruSeq® RNASample Preparation v2 Guide) at the PCR ...
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Why does one combine PCR and cloning as ways for amplification of sequences?
Why does one combine PCR and cloning as ways for amplification of sequences? Don't they produce the same result? I was reading the paper https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1864885/ and got ...
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1answer
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How do primers anneal to ssDNA?
In a PCR type protocol, the high temperature stage of the cycle will cause dsDNA to denature, as well as any annealed primers. At the lower temperature the denatured dsDNA will remain denatured. As ...
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In touchdown PCR, should the extension temperature match the annealing temperature in phase 1?
For TD PCR, it's recommended that the annealing temperature (T_a) be 10°C higher than the normal cycling T_a for the first 10 cycles or so, dropping 0.5 to 1.0°C/cycle. Should the extension ...
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2answers
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I did an agar gel electrophoresis but I could not see DNA, why? [closed]
After performing a PCR, I ran the products (700 bp) in an agar gel electrophoresis along with a Genedirex ladder (100 bp), but the transilluminator did not reveal them.
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PCR in different cell line
I developed a PCR cycle to amplify a 5.5 kb genomic DNA region in HEK cell line. When I tried to use the same protocol in A375 cell line I had no results but only smear. Can you help me?
Thanks in ...
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Issues with Mutant Allele Fraction Preparation
I am currently trying to prepare mutant allele fraction (MAF) solutions for an allele discrimination qPCR assay. I began with plasmids cloned with the different desired alleles. I amplified the the ...
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Gene cluster of interest not being amplified in PCR
In a lab, I currently have a sample of Rhizobium sp. NT-26. This bacteria is a chemolithoautotrophic arsenite-oxidizer, and I want to clone the arsenite oxidase genes into another bacteria strain in ...
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1answer
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Can Dpn1 digest hybrid PCR product with only one methylated strand?
In Site directed mutagenesis using PCR, after a cycle we obtain a hybrid molecule with one parental strand and other newly synthesized unmethylated strand. This is followed by Dpn1 digestion. Does ...
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Why both forward and reverse primer are added in asymmetric PCR?
If the purpose of Asymmetric PCR is to amplify single targeted strand of dsDNA then instead of adding just one primer for targeted strand why do we add both forward and reverse primer? Upon addition ...
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1answer
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Can PCR detect phosphorylated protein? [closed]
I'm trying to detect the level transcriptional protein inside cells. However, the cells contained both of this protein that is phosphorylated and not phosphorylated. Can RT-PCR detected the level of ...
