Multiplexing - Science method

I am new to CRISPR and we are trying multiplex genome editing in plants for 3 guideRNAs in a vector for a gene. We are following paper's Additional file 4 :Method S3 : Golden Gate cloning method for the assembly of 2-3 gRNAs. I have already designed my primers based on the instruction and got my Plasmids from Addgene pCBC-DT1T2 and pCBC-DT2T3. I am stuck in first step only which is PCR using these plasmids as templates. I checked the vectors by pcr --they looked fine to me but when I am running PCR with designed primers it is not working. I have used Platinum Superfi II green master mix with cycling conditions 98 -30 sec, 98C -10sec, 60C-10sec, 72C-30s, 72C - 5 mins hold for 30/ 35cycles and 4C hold. I am not sure if there is a problem in PCR conditions or primers since I don't know what should i used as positive control to clarify the problem

Please let me know any suggestion. IT would really help :)

I am using RNAscope Multiplex Fluorescence assay for RNA ISH, the protocol for fresh frozen tissue requests using a RNAscope probe dilution product to dilute the probe of interest from a 50x to a 1x working solution. A colleague of mine mentioned they use an off target probe to dilute their 50x. Thoughts on whether the negative control probe (a bacillus subtilis gene targeting probe) could be used as a probe diluent for a DRD1 mouse brain probe?

(trying to spend less while I optimize the RNAscope protocol for my tissue)

Is this toolkit only an extension for YTK (MoClo Yeast toolkit), does it assume that I have this kit and is based on YTK level 0 fragments?

Are there any regular YTK promoters or only the newly added inducible promoters?

The number of pores in the R.10.4.1 flow cell decreased significantly from +/- 1400 to 291 after nanopore sequencing with only 24 samples multiplexing. I used the SQK-RPB114-24 kit for processing the 24 samples as one library. Would anyone recommend anything about the protocol or to change something about it? Does anyone have about the same experience and what did you do to make it in some way better?

I am using Multiplex PCR, I want create an illustration like that for my primer ranges but I don't know how?

- How this multiplex primer ranges done?

- Any program?

- Is it for free or purchase?

I attached a photo of the published paper to be more clear.

Could the tissue quality/age be an issue ??

Hello,

I am working on a multiplexing EvaGreen-based dPCR assay and I was wondering what should be the minimum size differences between the amplicons to allow identification and quantification for each amplified gene?

I already tried with 4 amplicons in singleplex at 100bp, 200bp, 300bp, and 400bp, which was promising except for 400bp, which seems to be a bit much. I also tried other amplicon sizes at 60bp.

For now, I think I should test with only amplicons at 60bp, 100bp, and 200bp (to stay in the recommended size range of amplicons for dPCR) and maybe with 0.5X EvaGreen dye (to lower the fluorescence intensity and avoid exceeding the limit of the reader).

However, I have been working in dPCR for only a few months, so if any of you have advice, it would be welcome.

PS: I am also looking for which restriction enzymes could be used to fragment the gDNA non-randomly to improve the detection/quantification. Do you know which ones could be interesting to test?

Hi all, I was just wondering if anyone has experience with multiplexing a mouse monoclonal primary and a rat primary. I'm trying to multiplex by incubating them in the same well but was told by a colleague to research the literature and find out if anyone has multiplexed with the same mouse monoclonal ab that we're buying and a rat primary. Their concern was background staining due to the species being alike (their secondaries could bind to the wrong primaries). We're using a mouse-on-mouse blocking reagent from vector. Does anyone have any experience with this or any recommendations? Thank you!

I have done an experiment using luminex technique, measuring 46 analytes. but my sample size is less (N=6, for case and control each). However, i have put up the experimental set in duplicates. Can I use duplicates for the statistical analysis?

I am working on two viruses, when I do PCR for them separately they both showed on gel with their respective band sizes but when I multiplex them only one virus show different sizes and the other did not show at all.

MIMO

I am working on a multiplex immunoflurescence brain tissue slides

Hi,

I am doing three different Multiplex PCR, each with five pairs of primers amplifying regions of different genes. I tested the primers individually first and they all work fine. When i put them together for the Multiplex, one of the three primer mixes has the bands smear when I run the product on the gel (I am using 2% agarose gel in TBE1X, 150V for 1h). I have tried different primers concentration and also tested two different enzymes but the result is still the same. Do you have any suggestions on how to improve the multiplex?

Thanks.

Hello,

I am using RNAscope® Multiplex Fluorescent Reagent Kit v2 on 14 mikrometer thick brain sections mounted on super frost slides. Recently, I encountered a problem which was not an issue before. After applying RNAscope hydrogen peroxide, bubbles appear on the sections. As far as I can tell, they form also underneath the sections. As a result, I lose if not all, most of the sections on the slides. I would really appreciate if you can help me to identify the problem and eventually solve it.

Thank you very much!

Best,

Firdevs

For validation studies, I want to use RT-PCR for genes derived from sequencing data. For that I have to multiplex the reaction to see the expression for more than one gene. How many primers can be multiplexed together.

New to the qPCR methodology. I am looking for the most efficient and reproducible method to identify tissue-specific biomarkers using various tissues from a mouse model. I am planning to use Taqman multiplexing. Thank you!

I intend to perform cytokine measurements in rodent samples and would like to use the bead based cytokine multiplexing analysis, preferably using the Bio-PlexTM Pro Multiplex Immunoassays. If there is someone who has used this assay before, particularly for rodent serum samples, I would like to know how sensitive this assay is and what is the minimum sample size required to obtain reliable and possibly statistically significant quantification of serum cytokines.

SIW antenas equivalent circuit

I'm new to ELISA assays and have heard that multiplexing can be difficult. In your experience, what are some of the challenges that come with multiplexing? Is detecting up to 3 targets doable?

Dear colleagues,

We designed a transgenic cell line that gives us both a fluorescent and a luminescent output associated with different endpoints. Per se, we could culture and read out the fluo. signal on a black microtiter plate, lyse the cells, transfer the lysate to a white microtiter plate, and read out the lum. signal. However, I would like to increase the throughput by only using one of the mentioned plate types.

We are well aware of the caveats of using black or white plates regarding the fluo. or lum. measurement, as e.g. discussed here: https://se.promega.com/resources/pubhub/which-plates-to-choose-for-fluorescence-and-luminescence-measurements/

From your experience, which is the better plate type to use? Which one gives the better trade-off? Lum. is our primary signal, but as it is expected to have a better signal-to-noise ratio than the fluo. signal, I am inclined towards using the black plates.

Any kind of knowledge, experience, anecdotes, and trivia are much appreciated.

Thanks a lot.

Sebastian

I wonder if there is any available software to design multiplex qPCR assays ?

Please find below some of the details related to the study:

1. Total no of samples: 640

2. Organism genome size: 500 mb (in related species)

3. Restriction enzyme pair: SbfI and MSpI

4. Size selection fragments: 250-600 bp

5. Platform for sequencing: NovaSeq PE150 (120 G raw data per sample)

6. Multiplexing: 48 adapters X 12 PCR index

Looking forward to getting some help here

Hello,

I currently do multiplex immunostaining on mouse small intestine swissroll sections. We apply the protocol described by Adrien et Guillot al ( ). Images are acquired on different days and the alignment of images is done via DAPI channel. We encounter the problem that DAPI staining is either fading with each antibody stripping round (although we restain for it), or that the nucleus itself is not stable anymore and desintegrating (please find attached an image). This problem is only seen with us and adjacent research groups in the university. the protocol works perfectly for other universities/clinics. We tried to compare what is different in terms of reagents, fixation time, dapi, slides etc, it seems everything is similar between us and others! anyone has an idea what the problem could be? without a stable DAPI staining we unfortunately cannot do multiplex on our samples.

Worth to note that we use a histology facility that process our sampels (and adjacent research groups) and do the alcohol dehydration paraffin embedding steps for us. We suspect that it could be that the processing time is too long and the tissue is loosing its integrity. We will try a shorter processing time soon. Thank you for your help and input.

Best,

Asmae

I am using the Tecan MagicPrep automated platform to prepare NGS libraries of PCR products from DNA extractions.

Despite its fairly recent release (April 2022), I am curious if anyone has attempted making multiplexed libraries with this machine? If so, how well did it work for you?

Multiplex IHC is based on multiple rounds of incubating/detaching antibodies (+6 ABs) to assess expression of several proteins on the same sample. However, I didn´t find so far an explanation of how the detaching rounds are not affecting other structures in the sample (other proteins), but only the antibody/antigen bond (which is covalent)?

I am looking for a device that will perform automated multiplex immunofluorescence in frozen tissue and fluorescently label 4-5 markers on a single slide at the same time. Does anyone know which devices are better in this regard and have experience with them?

Am working on a two-element MIMO antenna, I extracted the multiplexing efficiency of my proposed antenna the result I have, is doubting because the values of the efficiency am getting is negative please can someone help me on how to extract the multiplexing efficiency?

I need to run some multiplex ELISA with kits from Millipore (Milliplex) and ThermoFisher (Procarta). However, I have 156 samples to run, more than the theoretically 76 spots available for sample testing. I would like to know if it is possible to extend the kit uses for more than the 76 spots available. My question is because it is needed 50 beads counts to validate the assay, but using the kit in the regular way, the beads counts are around 150-200 after the acquisition. So I thought if it would be possible to split the reagents to run more samples. My only concern is regarding the detection antibody and streptavidin tubes since they would be used in a lower concentration than recommended in this scenario.

I am struggling to find some antibodies for a project where I am looking at multiplexed targets in human, formalin-fixed & paraffin-embedded tissue. The criteria for suitability comprise:

Does anyone have any recommendations for a particular company, or if they have a database of proven antibodies in FFPE?

Thanks

Dear researchers

I do use multiplex qPCR, I use 4 different templates, Among these samples, only one sample give me one good Peak in flouresent per cycle and ct, everything is the same but the type of template. Does anyone could help me with this kind of abnormal peaks.‌

The architectures are based on different implementation methods such as carry select adders, ripple carry adder, multiplexers, compressors.

I am wanting to use DNA from Peromyscus leucopus and run it using Nanopore's MinION. I am wanting to run the whole genome and look at methylation. If I wash the flow cell immediately after it is finished, how many genomes could I run on a single flow cell? If I multiplex the samples (whole genomes) how many could I do and get usable data from a single flow cell?

I need to input multiple input through waveguide designed in comsol. I can design a single input of wavelength. How can I input a multiplexed input?

We are preparing qPCR reactions and wanted to ask if anyone has used PerfeCTa® SYBR® Green SuperMix (VWR) in their qPCR reactions? If so, can this mastermix be used in multiplex reactions?

Clearing-enhanced 3D is a medium used in histology and is compatible with most immunostaining methods including multiplexing IHC. It can also be used to increase the transparency of full organs. Nevertheless, it may take a long time for the incubation process to take place. My question is how can you overcome some of the limitations associated with the use of Ce3D clearing medium?

Hi,

I just want to know how to connect three wired strain gauge (TML strain gauge, type FLA-5-11, Tokyo Sokki Kenkyujo Co., Ltd.) with Agilent 34901a 20 channels multiplexer. A photo of the strain gauge is attached. Thanks.

Ahmed.

I am think Multiplex is better in terms of optimization and cost but might not be better in terms of ease of doing/handling.

Currently we are using the hybridization stripes from Hain Lifesciences but I am curious if there is also a (good) Real-Time-PCR to differentiate between the members of the TBC complex.

I'm rather new to sequencing protocols and don't get the difference between different sample multiplexing approaches using in single-cell sequencing. I know you can add sample indices in the Illumina library construction step. Those allow running multiple samples together, right?

Then there's which allows multiplexing using the barcoded antibodies. In addition, https://github.com/statgen/demuxlet allows demultiplexing reads from multiple samples if sample genotypes are known. What I don't get is why can't just the Illumina indexing be used? What are the downsides?

Thanks in advance!

hello

I work on field spreaders in multiplex networks. I need a database to do more experiments. It would be better if the database is on social networks with fewer nodes and five layers.

best regards

morteza maleki

I ve already done a singleplex of a set of primers against its target gene (DNA template) and it is positive giving a band at the expected size, when i do a multiplex by adding 3 other set of primers the result i have is no band.

However i tried gradient annealing starting from 4 degrees below the original primer annealing temperature up to 4 more degrees in a pattern of 2 degrees difference.

so can you help?

I recently prepared a cDNA library from EpiNext CUT&RUN RNA m6A-Seq Kit. I used the two sequencing adapters included in the kit which were a universal primer (Primer-U) and an index primer (Primer-I) for a singleplexed library. I was wondering if there would be any problems of re-running amplification/cleanup with NEBNext Multiplex Oligos for Illumina to do multiplex sequencing?

Hi all - I am testing some probe/primers assays to see if I can multiplex one of my ref gene (PPIA) with a target gene '(Tas1R2).

This is a typical amplification curve obtained - I have diluted the ref gene assay 1:2 to reduce possible inhibitory competition with target gene.

Still the target gene multiplex amplification curve show a shoulder that I do not see in the singleplex amplification curve. Any idea of what causes this ? Dimers between assays ?

Many thanks for your suggestions !

I would like to know if anyone have used or using RNAscope Multiplex Fluorescent Reagent kit for doing insitu's on free floating 40 um thick sections and can share the protocol with me. How long do we need to postfix the tissue in 4% PFA? The protocol I have says 1 hr at 4C. Also, do we need to use the barrier pen at all?

I performed the multiplex cytokine bead array (Thermofisher Procartaplex 20 plex) for plasma samples. However, I forgot to mix the contents while loading the standard 1 in the first well (A1) of the 96-well plate. Due to this, the standard curve formed is a bit deviated because of the standard concentration value of A1. I have attached the image of the standard curve formed. Please suggest how do I analyze the data.

We are planning to target a locus we believe to be an enhancer by multiplex CRISPR-based targeting and the dCas9KRAB and dCas9VP64 systems.

Our final vector will include 4 different sgRNAs (expression driven by 4 different promoters) that will target the potential enhancer. They are designed to do so by "tiling" that enhancer. The dCas9KRAB and dCas9VP64 will act as the repressors or activators respectively. We would like to test the sgRNA's accuracy and specificity before committing to the official experiment. One obvious way it to simply use an active Cas9 with each individual sgRNA and testing their efficacy a using genome editing test kit.

I am interested in a potentially quicker and more robust approach.

its important to note that we are only hypothesizing that region is an enhancer (based on previous data we have) and so testing expression of our gene of interest is not a viable option to test sgRNA efficacy.

I am running gel electrophoresis to confirm multiple target genes from PCR (not multiplex). Gene sizes range from 1.4 kb to 1.8 kb and the annealing temperature is 59 C. The same PCR reaction conditions are used for all the genes. I am getting some light bands less than 100 bp. Are these primer multimeres or primer-dimer etc? Can somebody guide me why these bands appear and how to get rid of these?

One of my WT probe for KRAS detection is ROX-taqman-MGB: While mutational probes are FAM labelled. My WT Probe always signals for mutations as well. Whether it could be the issue with probe (already reported probe sequence, change is just dye; replaced with ROX) or ROX dye? Any suggestions or help would be appreciated, thanks!

Hi dear,

Do you have any idea of a commercial lab who measure the cytokines levels in tears by multiplex or flow cytometry?

Thank you

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?

Hello,

I need to multiplex 32 channels to 1. The signals come from 32 piezoelectric transducers (transducer datasheet: https://acs-international.com/downloads/S1803_Data_Sheet.pdf). The goal is to acquire the signals , regardless of whether there are delays between the different signals. It seems to me that using a 32:1 MUX is a good choice. I plan to drive the MUX selects (S0-S4) with a micro controller ( eg: Arduino). I will use one amplifier at the output of the MUX. The problem is that , the transducers (A0-A31) output low voltages of the order of (µV-mV). So I don't know which multiplexer to choose. Will any MUX be sensitive to low voltages and not distort the signal ? What are the important parameters I should take into account when selecting the multiplexer?

done

Thank you in advance

We are using Cobas z480 for multiplex RT-PCR using 3 taqman probes FAM/VIX/CY5.

Once the PCR finish we want to see in the amplification curve the three results for each sample at the same time. This is possible in LightCycler 480 SW. If it is, could you explain me how?

Another doubt about this system, is it possible to export in the same table the three results for each sample?

Is someone working with the CFX96Touch Real-TimePCR System (Biorad)?

How is your experience with this system? It is our first option for multiplexing qPCR systems because it is in our budget range price.

Other options are:

CFX96Touch Real-TimePCR (Biorad)

QuantStudio 3 (Thermo)

StepOnePlus (Thermo)

We are interested in at least 5 channels, robust performance, versatility, cost of reagents, and excellent customer support. What to think about them?

Thanks a lot in advance!

I'm searching for a disease network (encompassing multiple diseases) that includes metabolites. That is, something like the Human Disease Network (http://snap.stanford.edu/deepnetbio-ismb/ipynb/Human+Disease+Network.html) or the Multiplex Diseasome (https://github.com/manlius/MultiplexDiseasome) but with both metabolites and genes included. Is anyone aware of such a network?

We are doing expression analysis for Zonulin, Claudin-1 and GAPDH from stool sample. For this work, firstly we extract total mRNA from stool sample by RNAeasy Mini kit and then synthesize cDNA for Zonulin, Claudin-1 and GAPDH using reverse primer (for Zonulin, Claudin-1 & GAPDH) in a single tube by Revert aid cDNA synthesis kit. Then, in qPCR analysis we do PCR in a single well for each gene (not multiplexing) and get lower CT value (around 16). However, we find very higher CT value (more than 35 or even near to 40) when we synthesized cDNA for single gene. Is it possible or scientifically valid to synthesize cDNA by multiplexing these three reverse primers of these three genes in a single tube? Could you kindly give a suggestion about multiplexing during cDNA synthesis?

Could you please help me how to multiplex SCAR marker with 18s rRNA markers?

I have conducted multiplex primers optimization by changing the final concentration of 18s rRNA, as follow:

SCAR (500 nM) and 18s rRNA (300, 200, 100, 50 nM). However, the band of SCAR marker still did not appear. Only 18s rRNA band appeared. Please let me know if you have alternative solution for my problem. Thank you

Dear All,

I am going to use EvaGreen®/ Bio-Rad system to detect two genes using two pairs of primers.

The first pair was successfully detected previously using EvaGreen® master mix.

It was detected at thermal cycler annealing temperature 51 degrees with a 99 bp amplicon. However, I would like to design multiplex and detect two genes using the EvaGreen® master mix.

The second gene has a master mix designed from BioRad and the recommended temperature is 58 degrees with an amplicon of 195 bp amplicons.

How could I use the same well in ddPCR to detect both? Which Temperature should I use in my multiplex assay?

Thanks all

Best

Hello multiplex experts,

I am looking for a multiplex solution (4 markers or more per slide) to run immunophenotyping in joint tissue. I anticipate extremely strong autofluorescence tissue background, so fluorescent multiplex like Opal (TSA) might not be an optimal solution. Can you please advise a chromogenic kit for 4-plex for manual staining? Any comments on IHC in joint tissues will be very helpful.

Thanks,

Elena

I am hoping to stain whole mouse brain hemispheres with fluoro-jade C as a marker of degeneration. We are presently working to optimize this staining for tissue cleared with the LifeCanvas SHIELD tissue-clearing method, followed by light-sheet microscopy. Has anyone had success with this approach or similar? If so, was permanganate treatment necessary to reduce background? Finally, has anyone been able to multiplex with immunostaining?

Thanks in advance for your help!

Does anyone have any experience using multiplexed IHC methods in fresh frozen human tissue, ideally brain? We are currently trying to set up techniques for labelling of a-synuclein Lewy Body Pathology and Neuronal cells in fresh frozen human brain and although we've had some limited success with a protocol utilising an IHC DAB imaging for synuclein, the background we are getting from non-IHC based methods of neuronal counterstain (cresyl violet, H&E) is very high and making assessment difficult. These slides will be used for Laser Capture, so we are limited in what we are able to do in processing the slide. Ideally we want to remain with chromagenic detection and not use immunoflorescence.

We are interested in exploring a multiplexed IHC platform, such as the Immpress Duet Double staining HRP/AP Polymer Kit that vector provide. Does anyone have any experience with this kit or recommendation of other multiplex platforms for dual IHC staining in fresh frozen, unfixed human tissue (ideally brain).

I am trying to stain for human cancer tissue

Hello,

I am interested in measuring the difference of IFN type I levels in plasma samples. I understand that I need to do a multiple ELISA for that matter and I was wondering if there are any suggestions from the community?

Thank you!

Dear All,

I have set up a 5-plex qPCR. The multiplex works fine but the PCR efficiency doesn't look that great. I am throwing in 250ng human genomic DNA and I get Ct in range of 30-35 for all my products. Average Tm for my three sets of primers is 60 Degree. I am currently using a combined annealing and extension of 60 degree with a hot-start enzyme. I realized if I split it into lower annealing maybe around 57 degree and a bit higher extension around 62 degree, it should improvise the overall efficiency.

Any comments? I am using Smartcycler -II

Appreciate your help!

In OFDM  based multi carrier transmission, whether every carrier carries a vector of symbols or a single symbol. 

Thanking you. 

Dileep M D

I want to simulate programme in matlab using OOFDM( Optical orthogonal frequency division Multiplexing ) any one can help me for coding

Orthogonality division multiplexing(OFDM) is widely adopted in many RF-wireless communication systems such as LTE ,WIMAX etc.In optical communication,This method of multiplexing is demonstrated many times. But according to my knowledge still OFDM is not used in available optical networks.

I want to know are there any optical communication networks that use OFDM?.

If not what are the practical issues encountered with the implementation?.

I have already experience with "muxViz" tool and "multinet" package in R. They use some predefined layer like "Fruchterman-Reingold". After plotting the network, I need to change the position and color of a group of nodes, however these tools do not allow such changes. I wondering if there is any library like tkplot in igragh (which is an interactive visualization tool for single-layer networks).

In 5G Networks bandpass filters that can optimize bandwidth allocation by eliminating noise, side lobes and Intersymbol Interference (ISI) in Orthogonal Frequency Division Multiplexing (OFDM) systems.

Which Illumina-compatible sequencing kits are recommended for the preparation of multiplexed libraries that cover the ITS region of fungi?

The probe is correctly binding but there is no rise in fluorescence.

I am doing a multiplex stain for CD3 and CD4, using Opal520 for CD3 and Opal690 for CD4. I am following the multiplexing protocol by PerkinElmer.

Each Opal is 1:50 (recommended dilution by manufacturer and the dilution our lab works with for multiplexing), anti-CD3 is 1:400 and anti-CD4 1:500, as those have performed the best from all dilutions tested in a dilution range and were also frequently used by our lab for multiplexing before.

When I performed the multiplex stain, I have noticed that the numbers of CD4-positive cells detected with CD3&CD4 multiplex stain are lower than their numbers detected with a control single anti-CD4 stain. Due to the close proximity of the receptors, I assume the problem is caused by steric hindrance. I am thinking of diluting primary antibodies and/or Opals and see if it helps when I repeat the stain.

For those who have dealt with this problem before, how have you solved it? I would like to spend as little time on this as possible.

Preferably dyes that have a narrow emission range as I need to use it for multiplexing

More specifically, I have 7 genes of interest (including HKG) to study in 3 different conditions and I want to use specific primers for each gene. When preparing cDNA, I put all my reverse primers together for each condition (so 7 primers per tube, for 3 tubes of different mRNA samples). Then I performed qPCR on my samples with separate couples of primers in each well. Importantly, I should mention that the reverse primers used during cDNA synthesis all have a tag sequence in 3` (not found in my bacterial genome of interest) so they are partially identical. That tag sequence was then used as a template for an independent reverse primer used in the qPCR step (and thus common to all targets, contrarily to the forward primers that are specific). I also added a no RT control (cDNA samples prepared in the same way with 7 primers but no RT) to be sure that there is no non-specific amplification during qPCR. Is that all right or do you think I should do more controls? Or should I prepare the cDNA independently for each gene (but then I need the add a primer for the HKG anyway).

I am running a Multiplex, and I would like to know if someone has some good idea how to increase my gel quality. So I am using 3 primer set and different DNA. I always integrate my Water control as last (the one to the right, before the marker) and before there is a blank space, to see if there is any well jump from one to another pipetting. I see the bands quite well, but I have this "smear" around the band and I don't know how to get rid of it. For a PCR reaction I normally load 12.5 microL on the gel and let it run at 85-95 V (2% agarose gel, 200 mL). I use the following MasterMix (for 9 samples + 10% pipetting mistakes):

Buffer 40 (microL)

dNTPs 4

MgCl 8

Primers 5

(Dream)Taq 1

DNA 1

up to 200

For the concentration I use to dilute my primer stock 1:10 and keep it separately from the stock itself.

For the dNTPs I use d(A/G/C/T)TPs 2.5 mM

the rest is pretty standard and is used from the DreamTaq kit.

Is it possible that my MgCl/dNTPs ratio is not optimized?

I think this might be my problem, because my DNA is quite ok (quality is acceptable, around 1.6-1.8).

I´m designing probes for qPCR multiplex with Taqman chemistry for gene expression.

What they consider should be the ideal size (range) of PCR products?

Thanks a lot for your time.

Rolando.

Hi everybody,

I try to measure the telomere lenght by PCR method. First, I tried to use multiplex method. Now I try to measure them separately (telomere and single copy gene). But curves during PCR do not have 100 % shape and I miss "Plateu" phase. Because of this reasson, I tried higher concentration of DNA (isolated from PBMC) with the same primers concentration, but unfortunatelly I got only different Ct. I attach the PCR curves.

The secong problem is, that I have lower amount of telomere product than product of single copy gene (Ct about 22). When I take a look on melting courves, I really have only one product...

Does anybody have similar experience? Can somebody give me tips, how to improve the protocol?

I will be glad for any tips.