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

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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
35 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 ...
3
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1answer
41 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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39 views

Which DNA fragments do not have expected sizes on this gel electrophoresis?

The problem is such: After performing a PCR, the vector carrying the PCR fragment with two restriction enzymes (Nhe1 and Asc1). The DNA samples were then separated using agrose gel electrophoresis ...
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19 views

What are flagged primers?

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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43 views

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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31 views

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
33 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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2answers
83 views

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
43 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 ...
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1answer
41 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 so ...
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2answers
49 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
90 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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60 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 ...
4
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1answer
80 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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0answers
32 views

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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2answers
51 views

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
32 views

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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2answers
153 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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0answers
13 views

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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0answers
48 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
51 views

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
39 views

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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24 views

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
37 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 ...
4
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2answers
163 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 ...
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0answers
107 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 ...
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2answers
113 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 ...
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1answer
92 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 ...
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1answer
90 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 ...
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42 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 ...
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1answer
126 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 ...
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2answers
55 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 ...
2
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1answer
170 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 ...
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2answers
489 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 ...
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1answer
22 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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15 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 ...
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0answers
14 views

What is the Magnitude of Nonspecific Oligo Binding at Low Temperatures

I am working on designing a protocol to capture genomic sequencing bound oligos (the part in question is identical to primer binding of genomic DNA during a PCR reaction). I'm wondering if anyone can ...
3
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2answers
359 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, ...
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37 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
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1answer
43 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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71 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
68 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 ...
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15 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
87 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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58 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 ...
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21 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
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1answer
61 views

DNA polymerase in PCR (polymerase chain reaction)

Can the DNA polymerase in PCR (polymerase chain reaction) recognize both DNA and RNA for use them as template? I want to know is it possible if my primers bind to an contaminant RNA and then any DNA ...
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51 views

Primer Designing [closed]

I have a basic idea about the requirements in primer designing. Basically my knowledge is limited to theoretical knowledge and have no experience in actual primer designing. And also I have a basic ...
2
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0answers
36 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 ...