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

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2answers
266 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 (...
3
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0answers
103 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 ...
3
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0answers
48 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 ...
2
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0answers
52 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 ...
2
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0answers
33 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 ...
2
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0answers
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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0answers
41 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 ...
2
votes
0answers
66 views

Based on which criteria should i choose the control sample to calculate delta_delta_Ct

I want to calculate the gene expression of an experiment with the Delta delta Ct method. I have these results from two runs of a real time PCR (one for the GOI(Gene of Interest) and one for the ...
2
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0answers
31 views

How closely correlated is the level of an snRNA with the level of its corresponding snRNP?

Let's say I am growing cells under two conditions, and let's say I measure U1 snRNA levels in both using digital PCR. If the U1 snRNA level is reduced in one sample, how safe is it to say that U1 ...
1
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0answers
75 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 ...
1
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0answers
91 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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0answers
37 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 ...
1
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0answers
19 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 ...
1
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0answers
87 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?
1
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0answers
187 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
vote
0answers
69 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 ...
1
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0answers
167 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 ...
1
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0answers
95 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 ...
1
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0answers
62 views

Cannot conjugate Biotin-labeled DNA to Streptavidin-labeled solid surface

I have been trying to immobilize DNA by the bioconjugation of biotin and streptavidin, but I cannot get this work. I added EDC and streptavidin to COOH ...
1
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0answers
46 views

What is the suitable terminology to describe this study approach?

I need to know the correct term(s) which are usually used in the parlance of both biology and bioinformatics for this study approach: About 11 transcripts were investigated using qPCR for a number of ...
1
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0answers
33 views

condensed protocol for sequencing a portion of human DNA from buccal sample

Anyone have a short & sweet protocol for PCR amplifying a region of human DNA (chromosomal or mt, I don't care) extracted from a buccal sample: including validated primer sequences and preferred ...
1
vote
0answers
62 views

DNA fingerprinting

I would like to make my own DNA fingerprint - just for fun to have my "autoportrait":). I was looking around a bit and all the commercial kits you can have are very expensive. Can you suggest me a ...
1
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0answers
46 views

RT-qPCR of selected differential expressed genes: which dilutions in calibration curve?

I've seen in MIQE guidelines that calibration curves a needed when performing RT-qPCR. I'll use genomic DNA to perform these calibration curves. My question is that I don't know the range of ...
1
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0answers
2k views

Bacterial 16S rRNA PCR amplification with universal primers

I am doing an experiment to amplify the 16S rRNA gene from bacteria present in gut contents of fish. After extracting DNA, I perform one-step PCR with universal bacterial primers (27F, 1492R) and I ...
0
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0answers
30 views

Problems with housekeeping genes

I have tried several housekeeping genes to analise the relative expresion of a cytokine for measure the inflamatory local response in mice ears, all the housekeeping genes I have tried are not stable (...
0
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0answers
14 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 ...
0
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0answers
21 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.
0
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0answers
20 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 ...
0
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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 ...
0
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0answers
81 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 ...
0
votes
0answers
25 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 ...