Questions related to Real-Time PCR
Hi, I'm doing presence/absence experiments with a comercial kit qPCR. The software requires an IPC-blocking agent but the kit does not have. Is there any configuration that allows it without IPC blocking agent? Moreover, the presence/absence graph does not appear in the analysis.Who know how to fix this problem? Thanks.
I extracted RNA from flax seedlings to measure microRNA expression by stem-loop PCR. I have done this many times with the CTAB method and often the 260/280 ratio is good and around 2 but the 260/230 ratio is between 2.3 and 2.4. Is there a problem with this ratio for cDNA synthesis or real-time PCR? Does this ratio have to be between 1.8-2.2? Please guide me if you have experience in this field.
Hello, researchers! Do you have any protein extraction protocols or tips that I could use for extracting SARS-CoV-2 spike and nucleocapsid proteins? I already know that a lot of protocols are virus dependent and that people use salts.
Considering clinical samples are in swabs and in transport medium for COVID19 real-time PCR diagnostic too, should we do a pre-treatment or something, before starting the extraction? What do you suggest?
I appreciate the help right away :)
1. Is there any DETAILED algorithms/videos to design primers and probes for real-time PCR?
2. Do probes really give much accurate results and are easier to work with (troubleshooting)?
Will be grateful to any speculations on that point.
Have a great day 😊
I have carried out Real-Time PCR in Applied Biosystem 7500. I mistakenly used relative quantification option instead of absolute quantification option. So I cant get Ct Value. But I have typical curve. There is any possibility to calculate the Ct value using the same hyperbolic curve in same software. I have limit of reagents only. Plz help me.
I want to do an absolute real-time PCR for quantifying the expression of 2 different genes. is it necessary to consider a reference gene? what is its role?
All components are freeze-dried. We use AB7500.
compare the liquid state, the other channels' noise signal of freeze-dried components are very high when we only detect one channel
In my q-PCR results, I got negative delta Ct values for some genes (where the GOI expression is higher than the reference gene).
In this case, should I repeat the q-PCR runs for the GOI with diluted samples to obtain positive delta Ct? or is there any analysis method that is appropriate with such cases?
Please comment from your experience of handling all 3 machines.
I want to do the gene expression test using real-time PCR, but I'm struggling with choosing the optimal housekeeping gene for my experiment? Do you have any advice for me? Thanks for your help.
I carried out 10 technical replicates for determining LOD95%. Below is the result (concentration in copy/mL, how many replicate amplified, and its positive rate)
10107 (10/10, 100%)
7580 (7/10, 70%)
5054 (6/10, 60%)
2527 (3/10, 30%)
1011 (1/10, 10%)
The result for LOD95 using Probit analysis is higher than 10107 copy/mL.. it is weird since the last concentration that shows 100% amplification is 10107.. can anyone help why this happens?
I would like to compare expression of my gene of interest (GOI) in non-treated leaves, stems, roots of my plant. In that case I don't have any control sample here and it's not possible to have one for such analysis. Efficiencies of GOI and reference gene primers differs above 5% and the efficiencies are not near to 100%. In that case I can't use delta delta ct method. I could use Pfaffl formula, but I don't have control sample (calibrator). How can I analyze my qPCR data?
Does the Illumina Eco Real-time PCR system require calibration for HRM (SNP detection)? The system manual mentions 'no temperature shift or calibration required'. However, I need to be certain before designing my experiments.
Any help would be appreciated.
I am detecting HAV mRNA by real time PCR using an in house developed technique (one-step RT-qPCR using TaqMan chemistry). The PCR program consists of 45 cycles.
Because the purpose of the method is qualitative, we will calculate the cut off value by statistical methods assigning only positive/negative results to our measurements (we use international standards). To do that, I first need to decide up to which Cq I will judge a sample as positive. Is there one correct way to set the threshold line in EVERY run so that we can have a Cq value that discriminates positive from negative samples?
My question relates to the fact that sometimes, we observe late amplification in samples that are thought to be negative and we want to know how to draw the line to separate them from ttuly positive ones.
Can anyone tell me if NALC-NaOH is suitable for liquefaction of sputum for real-time pcr amplification of non TB pathogenic bacteria and virus.
Otherwise how to prepare NALC-NaOH or NALC PBS buffer 2%
I am using TaqMan probes for SNP genotyping on LightCycler 480. I am facing some difficulties in resolution of heterozygous genotypes. The software can not identify it as " both alleles" automatically. I am using Universal TaqMan master mix with UNG.
Can anyone suggest me if including UNG activation in cycling conditions will make any difference in fluorescence call of FAM or VIC dye?
Hi, I want to isolate protein and RNA from the same frozen cells in SOLD buffer( RLT buffer+ beta mercaptoethanol). I will use RNA in the Real-time PCR and use protein in Western-blot. Anyone can help me?
I want to do HRM analysis from melt curve data which was generated from real time qPCR instrument of Bio-Rad CFX96. Our lab did not provide the original software from Bio-Rad (Precision Melt Analysis Software). Now, I kinda stuck. Any suggestion for free software that could process Bio-Rad melt curve data for HRM analysis?
To dear Researchers,
I was analyzing a series of concentration for estimation of Real-Time PCR efficiency. The concentration was 1:10. I used MS-excel to evaluate Slope. The result of slope was -8 and it was consistence with that in Scatter plot equation. I also drew a line plot. In the line plot the slope was -3.2 and the efficiency was 100%. Also, there was no linearity in Scatter plot but there was a well fitted linearity (0.99) in line plot. I'm not sure what is the problem. Please help me figure it out.
I want to multiplex some reaction with dual label probes.
I check the probes on FAM, and HEX channel. When I change the dye for ROX channel I don't see any fluorescent. I ordered the probes from two different companies, and both doesn't work.
I want to mix 3 probes in my reaction for FAM, HEX and ROX channel
Other people in my lab have used NormFinder but I'm trying to find other options which are user friendly as well
I Know that RT-PCR is Reverse Transcription PCR when the starting material is RNA. Going for reverse transcription first (RNA to cDNA) and then it could be conventional PCR with the need for gel electrophoresis or it could be real-time PCR reading the results without electrophoresis.
qPCR measures the amplification or we can say quantification, so qPCR and real-time PCR are the same? qPCR can be RT-qPCR if RNA is starting genetic material in reaction? Please if someone could clarify all these things in simple manner, it would be helpful.
I am about to search for the expression of genes including p53 in methylated and non methylated HPV samples. Should I extract DNA from the methylated samples and use SYBR green and Real-Time PCR to detect any possible differences? I have already the DNA from the non-methylated samples and cannot do an RNA extraction from them.
To perform HRM analysis for genotyping on CFX96 machine for the first time, BioRad recommends to use HRM calibration kit along with Precision melt analysis software. I am just wondering whether simply using any good quality DNA binding dye along with standard PCR reagents will work or not?
I am exploring a number of SNPs, unfortunately although the test are ok, I am perplexed because my preliminary results are nearly all against what is reported in the literature.
To confirm my workings, I need to understand if I have done this correctly.
I am using prepared primers from TaqMan for a realtime PCR. This uses two dyes: VIC and FAM, each associated to one allele.
An example of a primer would be the following:
where the manaul stated that A should be VIC and G would be FAM.
Does this mean that those results with a low Ct on VIC and a high Ct on FAM would be A or G allele?
Hello everyone, we are acquiring a new molecular biology laboratory with the fundamental objective of identifying species of microalgae and cyanobacteria. Perhaps you could help us decide if it is worth buying a real-time pcr, since an endpoint pcr is sufficient for our objectives.
Recently I am using QuantStudio 3 RealTime PCR 0.1 ml 96 well FAST. I am running experiments of amplification and melting curve analysis by SYBER GREEN I chemistry to identify different genotypes for gene of interest in DNA samples. Is possible to run high resolution analysis, and so to visualize different graphs from the basic melt curve and Tm, in order to better discriminate the genotype of my samples in an easier way? I am using the software suggested by Thermofisher (QuantStudio Design & Analysis Software v1.5.1), do you know if it would be better downloading a different or a specific software instead?
I would be glad to have any suggestion from you
I performed real-time PCR on luteal cells from cows which were treated with various factors for a period of 6 hours. While for western blot, cells were stimulated with the same factors but for a period of 24 hours. Is it possible that a given factor inhibits the expression of the studied gene and increases their protein level? Is the opposite situation also possible, when there is an increase in gene expression, while in western blots a decreased level of protein is observed? Is the difference in times of influence of the given factor could be the main reason or or other mechanisms may cause such results?
High-Resolution Melting (HRM) analysis is a relatively new, post-PCR analysis method used to identify variations in nucleic acid sequences. The method is based on detecting small differences in PCR melting (dissociation) curves. It is enabled by improved dsDNA-binding dyes used in conjunction with real-time PCR instrumentation that has precise temperature ramp control and advanced data capture capabilities. Data are analyzed and manipulated using software designed specifically for HRM analysis.
I wanted to run HRM ( High Resolution Melting) using Corbett Rotor-Gene 6000 Real Time PCR Machine. When I opened the HRM folder of software , there was two option for running including HRM and HRM with pre-amplification.
What is the difference between them?
Currently I process a commercial Multiplex Realtime PCR kit on Quantstudio 5 system, but almost 20% of the runs has a distorted amplification plot (see pictures attached below).
Does anyone suffer this situation? What can I do to improve it?
I am trying to set up my standard curve for qPCR analysis, presence absence of D. septosporum, using a QuantStudio 5 real-time PCR system and associated software.
I am using a 5X dilution series with a multiplex mix of fungal specific primers with ROX probe and 18S control primers with TAMRA probe.
Unfortunately I have not been able to generate a suitable curve for the fungal specific. So far, I have ran a temperature gradient to optimise annealing temperature.
I have an acceptable curve for the 18S which says to me my dilutions are fine.
I have adapted the volume of sample in each well and have found the amount that works well for the 18S primer-probes.
and I have eliminated the passive reference dye as an issue.
My amplification plots look good and how you would expect them to, however the curves are all over the place.
At this point I am struggling to find where to go next, any advice or suggestions would be greatly appreciated
I am working on quantstudio 5 system with diagnostic test kit - AZF Microdeletions REALTIME PCR genotyping kit (master mix is aliquoted to every separated pcr tube, I only added taq polymerase, mineral oil and DNA for pcr preparation step)
After doing several tests, i found some abnormal amplification curves (heaved curve with a small rise at very end pcr cycles) (attachment picture).
Does anyone know what 's happened?
Besides, the threshold on the picture is set automatically by QS5 system, can I trust this threshold to determine whether my pcr target presents or not?
I am really appreciated for your help.
Hengzhi Shi , Minwei Li , Xiaocui Huang, Chaoqun Yao , Xueqiu Chen , Aifang Du , Yi Yang
- PMID: 32731054
- DOI: 10.1016/j.vetpar.2020.109193.
I'm working on a project to detect the deletion of exons. We are working with Realtime PCR and using Livak's method. But we don't have a positive control of the target gene, we only have a positive control of a different gene. They are on the same chromosome. Can I use gen A's exon for a positive control to detect a deletion of gen B's exon via Real-time PCR?
We think yes because they follow the same principles. But I don't see any other published paper that did the same way we do. If anyone who has done this before or knows that it's ok or not, please let me know.
Thank you in advance and hope you all have a nice day.
When we want to use Lanthanide-based DNA dyes (Eu, Tb)for time-resolved real-time PCR, the colors are limit. So how to develop more multi-color Lanthanide-based DNA dyes?
I do a lot of RT-PCR and always include a no-RT control (without reverse transcriptase) for each of my samples, so that I can rule out DNA contamination in my RNA samples. Unfortunately, the SuperScript III 1-step RT-PCR kit (with enzyme mix of SuperScript III reverse transcriptase and Platinum taq polymerase) does not come with enough buffer to allow for this, which is a shame as this kit is very expensive. I contacted Thermo and they told me that they cannot sell me spare tubes of buffer and also cannot give me the composition of their secret optimised reaction buffer. This is a shame, though I totally understand why they would want to maximise their profit margins by pretending that their kits contain enough buffer for 50 reactions when in reality, they don't.
This led me to wonder if it isn't possible to guess the buffer composition and try to make it myself. The one hint that is given on the product data sheet is that the 2X reaction buffer contains 0.4 mM of each dNTP and 2.4 mM MgSO4. I also know from the information given with the Platinum taq polymerase what the composition of that reaction buffer is. I therefore wonder if one couldn't make the 2X reaction buffer for the kit by simply making a buffer containing 120 mM Tris-SO4 (pH 8.9); 36 mM (NH4)2SO4; 2.4 mM MgSO4; and 0.4 mM of each dNTP (clearly the platinum taq polymerase is very sensitive to chloride ions and the SuperScript III reverse transcriptase requires magnesium ions). I just have this very strong feeling that if one were to make that buffer, it would work very well indeed with the SuperScript III one-step RT-PCR kit as a replacement for the 2X reaction buffer, and could be very useful for making sure that the one gets the most out of this very expensive kit. I'm not sure how Thermo would feel if someone were to try that and publicise it on the internet, but somehow I feel like that recipe would indeed work very well....
you all have helped me along very much so far. I have now encountered yet another challenge: I need to design primers for 15 proteins which cover more than one splice-variant of the gene in question (ideally all splice variants).
As i know so far, Ncbi_Nucleotide lists all known variants and i can pick a primer via the button "pick pimers" for each specific primer. However, findig a primer wich picks up a lot of different splice variants may be just a question of luck and a lot of trial and error in my eyes.
Is there any programm/website or loophole which could help me along?
Again, I will deeply appreciate your answers.
Greetings from Munich
Ps: Because of this community I could actually get my project going last week. To all members: THANK YOU SOOO MUCH!
I am equipping a new molecular biology/microbiology laboratory and I need to purchase a PCR and/or a real-time PCR machine. So I'm looking for suggestions.
Is there a new model that performs well and allows highly reproducible results every time?
Thanks ahead for your input!
When performing Viral RNA extractions I perform a negative extraction using water. This is then run as a negative control for my RT-PCR reaction. Recently this control has been showing up RNAse P positive. In addition, when I run the same extract negative sample in duplicate on my PCR plate, sometimes I get RNase P in only one of the duplicates. What is happening? It only ever happens in the Extraction Negative, it never occurs in my PCR negative control.
This issue is occurring for multiple technicians.
Hi dear fellows,
I would like to perform ChIP-PCR on the promoter sequences of genes, but I am not quite sure how to design primers for the promoter sequences. Can someone please help me?
My second question is: is it possible that the real-time PCR for these primers has to be performed in some particular way?
Thank you very much for help
We know that passive reference dye/ROX can help reduce variations of fluorescent values in samples.
When utilizing the Rotor Gene-Q for the real-time PCR experiment, usually, the SYBR Green master (No ROX) dye is used. Why don't we use ROX dye while using Rotor Gene-Q?
What is the main feature that makes this machine unique in comparison with other real-time PCR instruments, e.g., ABI thermocyclers in the use of Passive dye?
I need to co-culture MLNs cells from a mouse in 24 well plates with bacteria. I will seed the cells 5x10^5 cells/well with the presence of bacteria 5 to 10 times higher. Is it possible for me to get a good quantity and quality RNA from 5x10^5 MLNs cells?
Thank you very much!
Dear all, Recently we tried to compare mRNA expression levels of a gene via real-time PCR in 40 colorectal cancer clinical isolates and 10 normal mucosa samples.
Interestingly, a reasonable expression was determined in tumor samples (Cq: 25-31) while no amplification at all (No Cq) was observed for normal mucosa samples. All the samples were processed for RNA extraction, cDNA synthesis and confirmation for a reference gene at same time. Our equipment is also working fine considering its 96 wells.
Would like to hear expert opinion from this forum for possible reasons why a reasonable expression profile was there for all tumor samples, while no signal at all was observed for the normal mucosa samples in the performed real-time PCR experiment.
I would like to compare expression of my gene of interest (GOI) between non-treated leaves, stems and roots of my plant. In that case I don't have any control sample here.
I evaluated efficiencies of GOI and reference gene primers in all of types of my material (leaves, stems, roots). The efficiencies of GOI vs reference gene primers are different in the tree groups of material and low (for leaves 64% vs 72%, for stems 66% vs 60%, for roots 70% vs 80%). I tried to redesign primers, but I obtained more or less the same results. My primers did not produce any dimers. What is worth mention, I isolated RNA with kit for plants rich in metabolites. Maybe the problem is the nature of my plant material, it contain some plant inhibitors.
Because of differences between primers efficiency I can't use delta delta ct method. I could use Pfaffl formula, but as I said I don't have control sample (calibrator). I could pick one of the material type and make it a control, but I have differences in primers efficiencies, and I think it would not be proper. I know the efficiencies are low, but it seems I have no choice here. Did you ever see such low efficiencies? What would you suggest to analyse my qPCR data? What could be a calibrator for my experiment?
Recently, I have been investigating of expression of Sall-4 in two different types cancers. Due to of acceptable melting peak of Beta Actin(and Cq=17), I am sure the quality of cDNA is pretty good.
I would like to know how to generate a dendrogram from repetitive element PCR profile. Please see the attached publication.
If somebody knows how to do that, please teach me.
For comparing the quality of two products, I used the same sample and primers to do qPCR, the results are as follows. As the figure shows, Tm values are a little different. I wonder if this can tell which of the two products is better, or whether it is judged by other parameters? Thank you.
I would be thankful if anyone could answer my question.
We are Analyzing the expression of a certain gene in our research.
We have extracted all the mRNAs in the cell and now I was wondering if we should Perform a PCR before doing real-time PCR? ( And Not for the purpose of quality check and control, But for the purpose of extra-Amplification)
wouldn't doing PCR on Our cDNA targets before real-time PCR result in a false increased Data about our gene amount?
I need help with the interpretation of why not obtain results in real-time PCR when I use Taq man SNP genotyping assay ID: rs 22756913 (IL17A) from Thermo fisher scientific
where the final volume reaction was10 microliter
Taq man SNP genotyping assay (20X) working was 0.3 microliter
DNA sample volume was 3 microliter with a concentration of 0.6 ng
the master mix was 5 microliter
nuclease-free water added to complete the total volume of 10 microliter
but when I use another SNP ID: rs 40401 from the same company (Thermo fisher ) with the same concentration and the volumes, I obtained results in real-time PCR
Why are CT values in the Realtime PCR hidden after the 35th cycle ?
Why are CT values in the Real-time PCR hidden after the 35th cycle ?
Where I noticed that the CT value had been positive and then, when the last five cycles approached, it disappeared so that it showed the value is unknown
I am using Applied Biosystems 7500 Real-Time PCR System for MTB kit , and there are two target the first one JOE is Intrnal Control (IC) and The second FAM which is the positive value C+ .
But I noticed the disappearance of the FAM value, which was CT 19 and 22 for some samples, and then it became indeterminate (unknown)
While the values of the JOE have never changed as shown in pictures
Please,if anyone know the reason for this situation
with thanks and respect
I will be happy for your help.
I'm doing real-time PCR with two primers called MT-TL1 and 18S, for mtDNA and houskeeping gene, and I got positive control on both NCT. I tried also different primers: ND1 and B-Globoin but also in them I'm getting positive ct value on ND1 gene (mitochondrial DNA gene), the b-globolin gene was good (undetectable).
I checked my reagents and they are good, there is no contamination. On these 2 last primers shouldn't be a primer-dimer problem.
I'm getting values of: 36.95, 35.59, 35.49
I used 10uM primer conc.
I would like to get rid of heparin in RNA extracted from heparinized plasma. Now I'm using heparinase II (Sigma H6512-10UN) dissolved in 100 uL of 100 mM sodium acetate pH 7.0. I used 1, 2 or 3 uL of heparinase with approximately 1 ug of RNA and incubated at 37 C for 1 or 2 hours continuing with 1 step RT-PCR but didn't success. Is there other protocol to get rid of heparin in RNA extracted from heparinized plasma by using heparinase?
I am working on a project involving detecting closely related fish species from environmental samples. I have 3 closely related species whose sequence similarities are such that it will be very difficult to design 3 separate qPCR assays that will distinguish them from one another without cross amplification, or at least cause reduction in reaction efficiency in mixed species samples due primer competition.
I was thinking of an alternative approach involving qPCR multiplexing, but do not have enough experience with qPCR chemistries to know if it will work.
What I am thinking is this: designing a multiplex qPCR assay with 1 conserved primer set that will amplify all 3 species, along with 3 separate probes that are species specific.
Is this something that would be possible? It seems like in general, multiplex reactions are composed of 3 different primer/probe sets that target non-overlapping regions?
Any thoughts would be helpful
I want to add some barcode sequences to primers then PCR them from different samples. Just want to know if there are any principles behind the barcode? I mean the length or the sequence features.
I know there are some commercial barcodes. But I need to add barcode to primers not the PCR product.
Hope for your reply!
A baby with congenital cmv infection tested positive using realtime pcr with 1000+ copies/ml of DNA. Further doctors ask for po65 assay why?? How do they go for treatment??
the concentration of my plasmid DNA is 126ng. i want to do real-time PCR taqman probe detection......i want to do serial dilution that should start from 10e8 copy number....so can anybody tell me how to dilute it (how much plasmid DNA and ddwater should be added to make it 10e8). i have attached the calculated values in the attachments. thank you
There are various methods of determining amplification efficiency as summarized under number 1 and 2 and in the bullets below:
1. From calibration curve slope as determined by:
· Fit-point method;
· Second derivative maximum for the 4 parametric logistical model;
2. From single amplification plots using algorithms like:
· Mid-value point regression — also known as data analysis for real-time PCR or DART-PCR;
· Window-of linearity algorithm or LinREG PCR; and
· Noise-resistant iterative nonlinear regression or RealTime PCR Miner.
So, I would like to know the most rigorous and classical method examines the slope of a calibration curve?
I wanted to run RT-PCR using onestep real-time PCR systems and SYBR green (ThermoFischer) to check the gene expression in bacteria. I wanted to directly use 50 ng of DNA samples extracted from bacteria. The Master-mix includes DNA template, DEPC water, forward and reverse primers. The reverse transcriptase enzyme was excluded since i'm not using RNA as a template. I wanted to know whether my protocol is wright?
Looking forward to all.
Thanks and regards,
Is it necessary to perform RT-PCR analysis of genes that show an increase or decrease? Or is it just for control purposes in RNA-Seq Analysis?
I want to use ddCt to analyze my qPCR results, however I am not exactly sure on how to proceed and when to average the replicates. Also, can we use ddCt when having more than 1 factor (e.g. treatment dose and treatment time)?
For example, I performed a cell culture experiment. I did 3 replicates (one each month during three month). I had two treatment times and three treatment doses (+ untreated control).
For each replicate experiments, cells were treated for two hours, or for 24hours, with a concentration of 0M (control), 10-7M, 10-6M or 10-5M. Cells were harvested, and using qRT-PCR I analyzed the expression of target genes + 3 reference genes.
In this experiment I want to see 2 things :
1. If gene expression is different in treated cells vs untreated control.
2. If gene expression is different according to duration of treatment (2h vs 24h).
I am not sure about how to correctly use ddCt and how to compare my two factors.... Should I first take the 2h experiment and calculated ddCt for treated vs untreated and than do the same for the 24h ? Then how should I compare times (2h vs 24h) using the ddCT ? Also, should I right away average my Ct values of the 3 replicates ?
I've attached my excel file with my ddCT calculations.
I really want to understand the rationale behind it..!
Thanks for your help
I already performed my quantitative PCR (q RT-PCR).
I have a lot of samples in my experiment and so they didn't fit all in the same rotor/plate (RotorGene - Qiagen). Therefore, I used a standard DNA sample on all rotors to normalize.
How do I calculate the normalization between rotors for the same gene?
I'm thinking to use the average of the DNA Cts, them correct in each rotor using the respective Ct value.
E.g: For gene ABC if I have 5 rotors/plates. The DNA Cts of each are 19, 20, 20.5, 21, 19. The average is 19.9. Then I go to the first rotor samples and add 0.9 to the Ct of each sample (19.9 - 19).
I don't know if I was clear in what I mean..
Any help is helpful :)
I am performing a qPCR standardization of some MGB probes. I have tried just a few times but I don't want to waste reagents, every time I tried the assay, there is a jagging line.
I am thinking that could be that the Reaction mix is not enough for the amplification or maybe I am doing wrongly the setup of the machine.
Do the MGB probes need an especial or different reaction mix?
These are my conditions:
Real-time PCR System: One step Plus - Applied Biosystems
Probe1: [FAM] GGG TTT AAA GGG [MGB]
Probe2: [CY5] GTC AAA TCA TCA TGC C [MGB]
1F: GTCAGCTCGTGTCGTGA 1R: CCATTGTAGCACGTGTGT 2F: AGCAGCCGCGGTAAT 2R: CTAAGCATTTCACCGCTA
Taqman universal master mix (applied biosystems): 12.5 ul
Primers 10uM: 0.5 ul
Probe 10 uM: 0.31 ul
Sample DNA (5ng/ul): 5 ul
PCR conditions (As reference article indicated):
50°C (2 min)
95°C (15 min)
60°C (1 min)
I tried to scale down the component in reaction (from 20 µL to 10 µL using Bio-Rad Universal SYBR Green) for saving material. I wonder if I can get precise results or not with a down-scaled reaction because the RealTime PCR machine in my lab was calibrated for using 20 µL of PCR reaction.
Thanks a lot for any advice/suggestions.
P/S. For testing, I prepared 45 µL of master-mix, then separated it into 3 tubes to make 20 µL, 13 µL, and 10 µL of reaction. The result shows a little bit different in Ct and quite big different in RFU.
I still get Non-specific products & multi-peak Melt Curves despite changing the annealing temperature of my designed primers several times (58C, 59C, 60C, 61C, 62C, 63.7C & 65C).
I've also changed other parameters like the concentrations of DNA & primers, however, i still get these Non-specific products & multi-peak Melt Curves. Are there other factors that i can change to get rid of them?
Thanks in advance.
I tried to run a qPCR assay using BioRad MyIQ qPCR machine but this machine failed to start run when I press "Run" button on the IQ5 software and showed the message which I copied below.
“Required background factor data not found. Perform background well factor collection for current seal (film), vessel (plates), base unit (yyyyyyyyyyyyy) and camera(569ER1245)”. Herewith, I attached the screen shot of the message.
This machine was purchased 5 years back. I used SYBR 1 for my experiment.
Much appreciated if anyone suggests me what I should do to sort this problem out.
We are intending to purchase a Real-Time PCR.
Which instrument performs better? Whether to purchase Peltier based model or rotor based model?
I have extracted total RNA from Lactobacilli and treated the samples with DNase I. When checking the -RT on qPCR, there was always gDNA contamination (appeared at cycle 33 or 35, automatic threshold) although the DNase incubation time was extended (normally 20 min, I extended 40, 50, 60, 70, and 80 min) either the enzyme concentration was increased (20%). Somebody said that it is hard to remove gDNA totally but it is still okay if the signal from gDNA does not appear before 30 Ct. I wonder if it is true or not, and would like to ask everyone for opinions about this issue.
The image shows up the result of undiluted -RT RNA sample (with and without DNase treatment). The NTC shows no amplification. With this result, can I proceed with my qPCR for relative quantification, or it need further DNase treatment?
Recently we decide to evaluate the miRNA expression levels in saliva sample as diagnostic biomarker of cancer .
We used TRIzol extraction methods but the CT of real-time PCR for U6 gene was about 31 .
How can i optimize the extraction protocol?
Our lab just obtained some 5XFAD Het mice that we plan to use. Our plan is to use WT, Hets, and Homozygous animals and was wondering if anyone had a better way to genotype them besides using real-time PCR (which is really expensive). Anyone has any primers suggestions that we can use for normal PCR?
I performed real-time PCR using an Applied biosystems machine. The double delta Ct values ( according to the machine) for the gene of interest after certain treatments were given as -5.12454; -4.93158 ; -9.11017. Does this tell me about the fold change in the expression of this gene? If Yes, then does this mean there is negative fold change or a positive fold change in the expression of my gene of interest?
Can we use money detector with UV lamp to detect fluoresceins such as FAM or FITC? For example product from Real-Time PCR with FAM or FITC probe?
For example we have a bacterium containing two plasmids, one of which carries a target gene. Is it possible to calculate the number of copies of this particular plasmid performing real-time PCR analysis of the target gene?
I am doing RNA immunoprecipitation, using the RNA TRAP technology, with which you can capture actively translating ribosomes with the L10 ribosomal subunit fused with GFP, using GFP antibody coated magnetic beads. Right now I'm testing it on HEK293T cells transfected with L10-GFP and I am having trouble finding good reference genes / controls for qPCR.
GADPH & b-actin seem to be differentially expressed between the IP & the unbound (the supernatant of the IP) fractions, which result in very different & unreliable results when I look at enrichment of GFP between IP & unbound. For example, b-actin sometimes shows Ct value difference of 5 between IP & unbound fraction, of the sample.. Actually b-actin seems to be 'enriched' in the IP sample (so I suspect it binds to the beads..)
What do you suggest I do? Should I compare with the input sample? n
I already get SNP candidates for our target traits, then I would like use Real-time PCR based genotyping platform to check F1 population.
For establishing the marker, the SNP flanking sequences are needed.
Does any one know?
Thank you very much
I have 2 different pairs of Primers, the annealing temperature of the 'reference pair' is 54C, while the temperature of the other 'test pair' is 60C.
I'm running my experiment on the device of Applied Biosystems '7500 Real-Time PCR System'. Thanks in advance.
I am doing realtime PCR for a known mutation.(a) Intra assay and (b)Inter assay validation are part of my experiments. According to my knowledge, Intra assay means running same sample mutliple times in one reaction and looking for the variations of results. And inter assay means running same sample in multiple different reactions and comparing the results. Now I have some queries:
- The sample that I used in Intra assay validation, should I be using that same sample for inter assay validation? Or I can use another sample?
- what is the minimum number of reactions for each assay validation? Like how many copies of sample 'x' should i have in a reaction of my intra assay validation.
- What is the minimum acceptable number of reactions for each sample in Inter assay validation.?
I hope my question is clear.
I am extracting a total RNA from rat brain samples for real-time PCR studies, but unfortunately, there is DNA contamination in gel electrophoresis. I tried to remove it and I failed.
By the way, I prepare to don't use DNAse treatment. (maybe the last option)
which part may cause this problem you think?