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Questions related to Real-Time PCR
Hello everybody,I am optimizing transporter protein primers of coleus with actin(250bp). I used normal qPCR conditions for the optimization with annealing at 55oC and cycles 55. Most of primers...
01 January 2017 7,224 16 View
I'm going to expose cells to hypoxic conditions, in order to establish the function of a synthetic promoter containing HRE's. For control sake, I want to see if cells actually developed hypoxic...
01 January 2017 6,865 4 View
Hi guys HCD test is a common test for quality control of recombinant protein in order to investigation of residual Host Cell DNA as a contamination in drugs. Considering 16srRNA repeats in E.coli...
01 January 2017 3,423 0 View
Hi everyone, I want to design a protocol to detect viral RNA of Hepatitis C virus though real time PCR with probes. I used to use a regular qPCR with primers against the 5'UTR region (most...
01 January 2017 742 0 View
Hi, I would like to get any inputs on determining the lowest viral detection limits using real time PCR. My initial trials have failed to give an amplification plot in the software, but I see...
01 January 2017 2,050 6 View
will large size 1,8kb of PCR affects ligation with 5.3kb of plasmid? I have repeated this three times; still, I'm not getting it, why it is not happening?
29 December 2016 4,746 5 View
We are going to do real time PCR with TaqMan SNP Genotyping assay to determine the genotypes of a population. However we donot have a positive control. I am wondering that is it possible to do...
22 December 2016 8,897 6 View
I am working with 16s rRNA , my amplicon size is 390 bp with 315f-806r , however I have got 350 bp , is it normal? please , if any one know answer , replay to me
12 December 2016 9,604 3 View
Hello! I'm trying to perform an optimization of qPCR for four genes of snail's nervous system (two isoforms of the same kinase and two housekeeping genes). I designed primers, checked them in...
12 December 2016 8,597 29 View
12 December 2016 9,646 6 View
Today, I set up the 1st PCR with 2.0µl templates following: 1, 5: negative control (water) 2, 6: Plasmid 1 (0.2ng/µl) 3, 7: Plasmid 2 (0.2ng/µl) 4, 8: Plasmid 3 (0.2ng/µl) Primers: gag-691F and...
12 December 2016 2,775 11 View
Hi I have a few questions about the extraction of RNA and RT-qPCR. I am aware that the real time PCR is normally run on more replicates of the same sample. I was wondering how it is better to...
12 December 2016 2,142 4 View
I was wondering if anyone has any advice on validating a gene knockout? I have a cell line which has my gene of interest knocked out with a 1bp insertion into an exon. I have tried qPCR which was...
12 December 2016 8,230 4 View
Hi, I can't understand well the use of the normalization for analysing reverse transcriptase real-time pcr data and why you need to use housekeeping genes. Actually my issue is that I can't...
12 December 2016 5,262 6 View
Hi all,Recently I keep getting this high molecular weight smear in my PCR, and it is present in negative control.The smearing is only observed when no amplicon is obtained? Makes me think there...
12 December 2016 9,350 11 View
I want to standardize RT-PCR for various microbial groups. Is it possible to do so using metagenomic DNA which might contain the same groups? Or do I need to use DNA extracted from an axenic...
12 December 2016 9,383 5 View
12 December 2016 4,961 6 View
What are the best methods for nasopharyngeal swab processing for viral q-PCR detection? After collection of sample and cut off the swab tip in the UTM then vigorously agitate swabs in medium for...
12 December 2016 7,177 2 View
about Real time PCR and positive control\ plasmid DNA how i can know the "'gene copy number" according to the Concentration of the plasmid DNA for using as positive control on Q Real Time PCR...
12 December 2016 2,305 1 View
before we doing the relative quantitative real-time PCR, we should generate a standard curve. when we are generating the standard curve what should we pay attention to to make your curve more...
12 December 2016 2,660 7 View
What is the significant fold increase for delta delta ct analysis for a prokaryotic gene. Is it above 2 fold? If so, have any one have got above two fold increase in any prokaryotic gene? Iam...
12 December 2016 5,488 4 View
I have been using genomic dna for the generation of standard curve of qpcr and I am getting very poor pcr amplification efficiency.
12 December 2016 297 3 View
I am using 5' RLM RACE to validate the targets of certain miRNAs in plants. I need to access the cleavage of particular targets among salt-resistant and salt-sensitive samples. Is it possible to...
12 December 2016 9,550 2 View
04 December 2016 7,064 2 View