Questions tagged [pcr]

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

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56 views

Choosing PCR conditions

How does one choose PCR conditions? Does it depend on the taxon, on the DNA concentration, on the primers or anything else?
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How is 16s rRNA used to identify unknown bacteria?

How is 16s rRNA used to identify unknown bacteria? With PCR the 16S rRNA is amplified and sequenced, but how can you identify the bacteria with this 'piece of knowledge' if the 16S rRNA is conserved ...
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58 views

Same primers and different melting peaks for two samples (~1°C appart)

In the figure, you can see that melting peaks differ by ~1°C at each sample. Should I be worried?
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1answer
168 views

Do all microRNA isoforms need to be known and sequenced to obtain microRNA expression?

From what I understand, microRNA are short "hairpin" shaped nc structures. When sequencing and counting miRNA, isoform miRNA are "found" which are subsections of varying length and possibly with some ...
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127 views

Principle of 16 S rRNA technique for bacteria identification [closed]

Why do we isolate DNA not RNA in 16 S rRNA technique for bacteria identification?
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1answer
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Is a Linker sequence mandatory in primers for PCR Cloning?

I have designed the primers for PCR cloning, but i was not getting any colonies after the ligation. One of our colleagues said, that the restriction digestion might not be working as there is no ...
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SRAP marker sequences

I am looking into SRAP markers and I have realized I am not sure if I need a reverse complement of the sequence given in the article or they are "ready to use" primers?
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0answers
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PCR limits of detection calculate method

Is any standard method for use to gain PCR sensivity or limit of detection? my mean is not for RT-PCR or Real-Time or any other kind and just simple PCR. After making PCR product free of dNTP and ...
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Is Melanin a PCR Inhibitor?

I have been growing B16F10 Mouse Melanoma cells. I need to extract the genomic DNA and do PCR to amplify a specific region. However, no matter what temperature or magnesium concentration I use, I have ...
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1answer
185 views

Why do we need to replicate incomplete DNA fragments by PCR from a crime scene?

If the DNA from a crime scene is damaged, why would it be helpful to replicate it by PCR (polymerase chain reaction)? Even if we get billions of those copies, it is still incomplete, isn't it? How ...
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house keeping gene variation under treatment

I am injecting BCG in mice ears to measure the local inflamation response in the infection side, one ear in the same mouse is kept as control by injecting PBS, in the other one, BCG is injected. When ...
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2answers
17k views

If we add only 1 primer in PCR [closed]

What would happen if we add only one primer, say forward primer, to PCR? (Image Credits: Wikipedia) As it is clear from the image that we need both forward and reverse primers to get it working (...
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1answer
33 views

Validation by PCR of a lowly expressed gene in microarray

I am interested in validating the precence of a gene (FETUB) whose expression is in the lower quartile of the microarray (see figure in link). https://drive.google.com/open?id=...
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How to find suitable qRTPCR reference gene for a inflammatory response experiment?

I have tried several housekeeping genes – Hprt, β-actin and GAPDH, to analyze the relative expression of a cytokine for measuring the inflammatory local response in mice ears. However, all ...
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ssDNA library amplification

Assume we are given approx. 80,000 dsDNA fragments of 40 bps length each. I would like to amplify of each of the 80,000 fragments just one particular strand, such that I obtain amplified 80,000 ssDNA ...
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1answer
228 views

Is this modification of Livak's 2-delta delta CT method valid?

I have done a modification to include qPCR efficiency in the calculations: In a given gene I calculate the fold changes relative to an arbitrary sample. That sample will be one and the rest will vary ...
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1answer
758 views

Why would you mix polymerases in PCR?

I was reading a manual for kit that requires you to amplify DNA by PCR before analyzing it in the kit. The manual suggests mixing proofreading and non-proofreading polymerases: For PCR products ...
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2answers
690 views

Separation of smaller DNA fragments and larger fragments without using gel electrophoresis?

For an upcoming experiment I would have two kinds of DNA fragments in a sample after a particular reaction step: First there would be fragments of a length of approx. 170 bps and then there would be ...
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221 views

Choice of primers for PCR

This exercise was given by my professor but I am struggling to understand the solution. A PCR is performed on the following sequence (in order to replicate the chain and thus have a greater quantity ...
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1answer
165 views

qPCR: do both primers have to contain a GC-clamp in order to be effective?

I'm developing software which, among other things, designs primers pairs (forward and reverse) for qPCR. In my research on various properties, I read about GC-clamps and I understand the basics of it ...
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1answer
167 views

Questions on adding a protein to a DNA library [closed]

Two questions regarding finding the DNA sequence of a amino acid sequence (AA): 1) If you are able to find out the mRNA sequence of an AA, then don't you automatically know the DNA sequence? 2) ...
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1answer
1k views

Why does Taq polymerase add 3' adenine overhangs?

Is there a mechanism for the preference of Taq polymerase to add a non-templated 3' adenine (overhang) instead of other bases?
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1answer
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Pooling for qPCR

I am comparing miRNA expression levels in 3 different groups but I am low on money and time. I have to get some preliminary results to get the actual research going so I decided to pool my samples and ...
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1answer
991 views

What happens if both forward and reverse primers have same Tm?

One of the key rules in primer designing is that Tm (melting temperature) of forward and reverse primers should be in the range of ±5°C. For example, if ...
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2answers
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Good pipetting technique?

Good pipetting technique is essential for many biologists, but it can be hard to get right. When I take 1 µl of liquid using a micropipette, I seem to always take less than 1 µl, and that amount is ...
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How to solve the problem of 2 melt peaks in real time PCR?

I am facing a problem in my real time experiment. After standardizing the annealing temperature by semi-quantitative PCR, I did real time for my gene of interest. In one of my samples, I got 2 peaks ...
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1answer
837 views

Purifying large amounts of PCR product

I have had trouble purifying very large quantities of pure PCR product. We are using these as templates for the reconstitution of nucleosomes and I require hundreds of micrograms to milligrams of ...
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1answer
556 views

Why are sequencing reads shorter than PCR products?

I have desinged and tested primers for RT-PCR, then purified the PCR products from the gels and send them to sequence at GATC (SUPREMERUN) using the forward primers. After blasting the reads to ...
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What are flagged primers? [closed]

I'm interested in amplifying a sequence for further use with Gibson Assembly. I want to create overhang regions in my DNA fragment so there would be complementarity to the plasmid I'm trying to insert ...
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Why do PCR manufacturers recommend assembling the reaction mix on ice?

Many PCR manufacturers recommend preparing the reaction mix for the reaction on ice. For example, here is NEB's recommendation for their Q5 polymerase. Other manufacturers also include similar steps: ...
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PCR that worked previously is now only showing primer dimers and a smear on gel

PCR amplification of a promoter sequence for gel extraction worked beautifully using Phusion HF enzyme with GC (higher error but less picky) buffer. However, DNA concentration from the gel extraction ...
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1answer
63 views

what benefits does lab procedure Realtime PCR have in gene silencing experiments? [closed]

RT-PCR performed in gene silencing but mechanistically what are the benefits of this procedure?
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In Sanger sequencing, why do we resort to cloning? Why doesn't PCR suffice?

I understand that in Sanger sequencing we can clone our fragments with the help of e.g. bacteria to make multiple copies of our fragments for further analysis. I also understand cloning can be a ...
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1answer
2k views

Difference between PCR for linear template and a plasmid?

I believe PCR can be conducted both on a linear template and a plasmid, and I was wondering how these procedures differ in what enzymes are used, how the enzymes work on the template, primers used etc....
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1answer
4k views

Why do you need primers in PCR? [duplicate]

I have read that DNA polymerase requires a primer to bind to the DNA, but I am confused as to why this is the case. When DNA undergoes replication in the cell, there are no primers in the nucleus. Why ...
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3answers
612 views

Getting PCR amplification at annealing higher than Tm!

I am amplifying a gene where in a gradient pcr i am getting amplification at an annealing temperature about 5 degrees (67) higher than Tm (62.5)? What is wrong here? Also, I am getting a very strong ...
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2answers
1k views

Alternatives to PCR

PCR uses cycles of heating and cooling to denature the strands, calling for special thermostable DNA polymerases. In a cell, during replication, Helicase unwinds the DNA without the requirement of ...
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348 views

What would be the shortest and optimal method of extracting human cells for PCR? Is there a colony PCR like protocol for human cells?

I am trying to devise a quick method to extract genomic DNA from human cells for a PCR. I first collected cells by centrifuging saline mouthwash (0.9% NaCl) and extracted genomic DNA using kit ...
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1answer
641 views

Freezing after restriction digest

Brief question: I have performed a double digest with heat inactivated restriction enzymes with no star activity. I usually purify the DNA (PCR inserts and linearised vector) using a kit or gel ...
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What evidence is there that common PCR DNA purification kits can isolate DNA from pathogens engulfed in macrophages?

I have previously asked a more general question about how PCR sequencing reaches all possible DNA targets here. I want to ask more specifically now: what evidence is there that common PCR DNA ...
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3answers
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Does common PCR amplify genes regardless of what cells / barriers they are in?

I have some understanding of how PCR testing works. What I have always been wondering: how can we be sure that a primer reacts with the targeted gene(s) regardless of where¹ the genes are inside a ...
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1answer
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Conjugated deoxyribonucleotides

I'm currently learning about using PCR techniques to make fluorescently labelled DNA probes, and the textbook mentions "conjugated deoxyribonucleotides" Can someone explain what these are? Nothing ...
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1k views

Polymerase Chain Reaction Questions

First question is about why we use primers in PCR. It requires two reasons. I know only one reason though. It is so that DNA polymerase can attach to primer and make a copy of nucleotide from there. ...
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Using Q solution with ready made MasterMix

I am exploring the possibility of using Q solution (5x) to get rid of non specific bands in PCR. I mostly use a MasterMix and not separate aliquots of dNTPs, Taq, buffer etc. In principle, adding Q ...
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471 views

What is the meaning of oIMR

In reference to genotyping primers, often some are labeling with 'oIMR'. It seems that this often refers to internal positive control primers, but I'm not sure. What does this stand for?
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2answers
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Troubleshooting PCR Steps [closed]

I am troubleshooting a PCR reaction that gives me no product, and there are a lot of different approaches that I can find in books and just in google searching (things like running a gradient, ...
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2answers
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Is it possible to amplify every single piece of DNA through PCR?

Is there a way to perform non-specific PCR amplification for the purpose of amplifying every piece of DNA present?
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BSA for trouble shooting failed PCRs?

I am using PCR to amplify a 1000bp region of the CytB mt gene. Some of the samples had not amplified and I wish to change the reaction components (or use additives like BSA) to attempt at getting ...
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1answer
78 views

Is there a good site or software to see if a primer pair spans an exon junction?

I am going to perform some RT-qPCR tests to validate an experiment.  I'm currently in the process of ordering primers, and I would like to get them from the Harvard Primer Bank since these have been ...
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2answers
2k views

How to Extract RNA and perform RT-qPCR from very few cells ( ~5,000)?

I am currently conducting an experiment which involves FACS-sorting a specific population of cells using an antibody of interest. In order to validate the type of cells I have collected using this ...