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Questions related to Real-Time PCR
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 2,146 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 8,245 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 5,272 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 9,364 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 9,392 5 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?
12 December 2016 4,970 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 7,182 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 2,313 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 2,669 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 5,502 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 305 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 9,555 2 View
04 December 2016 7,083 2 View
02 December 2016 8,402 6 View
01 December 2016 6,117 3 View
what is the prerequisite before you conduct relative quantative real-time PCR?
29 November 2016 5,359 3 View
Is best to prepare the RT-qPCR in one-step or two-step?
27 November 2016 9,694 8 View
Hey, I am doing RT PCR with both TAQ and SYBER primers. My normalizing genes are great, unify, and perfect. However OTC, even in HepG2 cell line, is not unify and raises in the same technical...
23 November 2016 6,282 3 View
I am doing gene expression analysis using rt-qPCR (iTaq™ Universal 2X SYBR® Green Supermix). I use Applied Biosystems™ QuantStudio™ 3 real-time PCR machine. I ran my control and treatment rat...
23 November 2016 2,017 4 View
Hello! I'm currently analysing some RT-qPCR data by Pfaffl method. My experiment consisted in: -the expression of 5 genes in two embryo stages (T4B and T7). I also used: - two biological...
15 November 2016 627 6 View
Hi Everyone, hope there is someone on the planet that could help met with advise on the villencre PCR. I do not get it to work. I am currently running a multiplex PCR with MYD88 as an control...
15 November 2016 4,250 2 View
RNA isolated from cell line and got a concentration of about ~ 300 ng / ul (purity of about 2.0. The RT-PCR must have a mix of 10 ul + 10 ul RNA wheel RNA - 1 ug. I performed the operation 1000ng...
14 November 2016 2,444 4 View
qPCR was done to a gene called 36B4, 20ng of DNA and forward primer with concentration 300 nM and reverse primer of 500nM.qPCR machine used is lightcycler 480. Program used 95 c for 10 minutes,...
11 November 2016 8,842 9 View
I know both buffer should contain Tris-HCl and KCl and MgCl2. Apart from this, what is the difference?
11 November 2016 4,430 6 View