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

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When designing primers how important is the GC clamp?

I'm designing a set of primers and reading about the principles of primer design one of which is: GC Clamp: The presence of G or C bases within the last five bases from the 3' end of primers (GC ...
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13 views

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
29 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 ...
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0answers
19 views

Effects of pulverizing liquid nitrogen frozen tissue by by pestle and mortar

Does RNA, DNA or protein molecule break during pulverizing (liquid nitrogen) frozen tissue by pestle and mortar. As I want to make cDNA for qPCR, this is very important to me to know this.
3
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1answer
28 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 ...
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2answers
41 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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18 views

General PCR is presenting with mysterious bands?

I have a small question. I recently performed a PCR reaction on FFPE DNA yielding a 125 BP fragment. However, a strange thing occured when I analyzed the results on our Fragment Analyzer. I got a ...
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2answers
48 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
32 views

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
37 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 ...
2
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1answer
61 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) ...
3
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1answer
71 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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259 views

How does LCR compare to Assembly PCR

The question pretty much explains itself. How do the two methods compare? I've always used Assembly PCR but the method is prone to mistakes and I'm curious how it compares to Ligase Chain Reaction (...
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2answers
91 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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0answers
15 views

How to prove that valerian acid binds to GABA A receptor [closed]

I would like to ask: what kind of method or tool that used to analyse a blood sample which contains valerian acid that binds to GABA-A receptor and cause sedative effect? Can i use real time pcr?
0
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1answer
70 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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0answers
16 views

Cloning Stragedy

Recently I am learning about TOPO cloning in a class. I have questions regarding to this: In the TOPO-TA cloning, they use topoisomerase I to cut the backbone of the vector so it will covalently bind ...
4
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1answer
149 views

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 ...
3
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0answers
88 views

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 ...
4
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1answer
76 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
75 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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2answers
2k views

Equation for accurate prediction of PCR yield

It is a cliche of freshman biology labs to point out that "every cycle of PCR doubles the DNA, so the yield will be $2^{cycles}$ times the template amount". However, if this were true, 1 ng of ...
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0answers
72 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 ...
3
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0answers
22 views

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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2answers
344 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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2answers
198 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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0answers
82 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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1answer
41 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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50 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 ...
0
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1answer
107 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....
0
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1answer
94 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 ...
2
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2answers
126 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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2answers
99 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
157 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
36 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
60 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
33 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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0answers
18 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
79 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
65 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
42 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?
2
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0answers
32 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 ...
1
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1answer
45 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 ...
10
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3answers
6k views

Why is the number of PCR cycles limited?

I've been told that the maximum number of cycles in PCR is between 20 and 30. Is this true, and what are the reasons for this limitation?
4
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2answers
335 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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2answers
265 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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0answers
172 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 ...
1
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1answer
112 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 ...
0
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1answer
162 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 ...