Real-time, real time, or realtime may refer to: Real time (media), a narrative technique in which the events depicted take place entirely within the span of the depiction, and at the same rate Real-time computing, the study of computer systems which are subject to a real-time constraint. 'It: Chapter Two' Adults Get Advice From the Young Cast Bill Hader, Jessica Chastain, Finn Wolfhard, Sophia Lillis and more describe playing the same characters years apart in the horror sequel.
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Quantitative PCR uses fluorescent dyes or probes to visualize the amplification of specific DNA sequences as it happens (i.e. In real time). The dyes or probes fluoresce when they bind to newly amplified DNA, and the amount of fluorescence emitted is proportional to the amount of DNA (or mRNA) present in the original sample.By detecting newly synthesized DNA during the exponential phase, qPCR is more sensitive and accurate than end-point PCR.
Hi, Yunes.I hope I can help you with your questions.1) It seems that a CT difference of more than 0.3 between replicates and/or a calibration curve R2 of less than 0.99 are unacceptable. How do we overcome the problem if that is the case?I don’t think they are unacceptable but they are not the best results you want to have.
So, you need to try to decrease the differences about your replicates to the minimal. If I did not get it wrong, Suzanne said up here in the text, o.9 at least.2) People use the blue and the yellow solution mixes. Blue is the Syber mix and yellow is the cDNA. Which one to go first and how to avoid contamination?Usually, the normal standard, was to distribute the SYBR mix first and then the cDNA.
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In this order, the chance to make air disturbance able to take the cDNA from one well to the other is less probable.Do we need to change pipette tips for every well? Or keep it for every set of replicates?In my humble opinion, You should avoid to pipette anything with the same tip to more than one well. I do change tips everytime.Finally, There is no such a thing as elementary questions. Your questions are the most important for the right flow of your work.
So, keep on doing all questions you have in order to eliminate them. Ok?Good luck! Master mixes are your friend. I find putting my template in the master mix and adding the primers to the plate give the best results. Make the biggest master mix possible at each stage (without template/primers; with template/primer). This is for two-step qPCR where I’ve made and stored my cDNA and use it for lots (and lots) of templates.My own personal method to reduce the number of tips without cross-contaminating is to pipette the primers into the wells with the plate back-to-front (top row facing you) and then swivel it around to add the master mix.
Seal with adhesive film and centrifuge at 3500 rpm for 30s to collect everything at the bottom.I use an external standard (lambda phage gDNA) to quantify by templates because my samples don’t have a stable internal reference to normalise to (the treatment affects primary, secondary and “housekeeping” metabolism). See papers by Rutledge on LRE for details. TE can actually inhibit your qPCR reaction. So what I do is store my stock standard (the most concentrated one) in TE for stability and then serially dilute my 1:10 standards in PCR grade H2O.
With a 1:10 dilution, as long as the standards you run on the plate are at least one dilution away from the stock, the TE should be dilute enough not to interfere with the reaction. Just be aware that your H2O diluted standards will be less stable than the stock and so you may want to make the dilutions fresh fairly often – as soon as you start to see increases in your Cts or if it has been a few months.Hope that helps!! Hello Suzzane,I am a beginner on RealTime PCR and am using Applied Biosystem SyberGreen mix for my PCR and I have the following questions:1) It seems that a CT difference of more than 0.3 between replicates and/or a calibration curve R2 of less than 0.99 are unacceptable. How do we overcome the problem if that is the case?2) People use the blue and the yellow solution mixes. Blue is the Syber mix and yellow is the cDNA. Which one to go first and how to avoid contamination?Do we need to change pipette tips for every well?
Or keep it for every set of replicates?Sorry for asking elementary questions.Yunes. Hi Marisa,You can store your primers at -80C but aliquot them to avoid multiple freeze thaws. I would recommend resuspending the concentrated stock of primer in a TE buffer so the little bit of EDTA will protect from DNase degradation and the buffer will protect from the acidity of water causing hydrolysis. You can aliquot this stock and put them at -20C. Then you make your dilutions of a working stock.
You can make the working stocks in just Tris buffer so the EDTA is diluted out (which is even more diluted once the primer is added to the PCR).The working stocks should also be aliquoted so that you do not freeze/thaw them more than a few times and also, in case they become contaminated with PCR product, you can easily solve the problem and not have to throw away the entire stock of primers.Storage at -20C is ok too and -80C is fine if you want to put them away for a long time without needing to get to them frequently. If you are in the middle of a project and need easy access to the primers, use -20C.
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