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

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6
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2answers
162 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
30 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
19 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
14 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
26 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
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0answers
29 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
51 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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0answers
11 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
41 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
45 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 ...
1
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0answers
36 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
47 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 ...
1
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0answers
40 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
27 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
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0answers
58 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
42 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
1k 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
votes
0answers
32 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 ...
0
votes
0answers
12 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 ...
0
votes
0answers
16 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 ...
0
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0answers
30 views

Real Time PCR Test Chemistry

I am working on building a real time PCR machine. Is there any chemistry set that I can purchase online to: Identify if PCR amplification was successfully done. Test fluorescence dyes that could ...