Realtime PCR plate cleaning

Realtime PCR plate cleaning

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Have anyone used realtime PCR, is that the plate for realtime PCR is one-time using or not ? Otherwise how to clean the plate after reaction to prevent wrong result in next time using ?

A possible solution is to use the DNA ZAP solution, which I know many people used in adjacent labs and were happy with for cleaning their DNA free workbench. The product sheet states

When 500 ng of template DNA is dried down in a PCR tube, it is rendered unamplifiable upon treatment with DNA ZAP solutions. Additional experiments demonstrate that the DNA is degraded down to free nucleotides.

Please note that I'm not promoting any specific product here and you can purchase any similar product/technology for your particular application. Obviously the best recommendation is to use new plates for each reaction but this/or a similar product can be an option too although the risk of DNA contamination in a PCR based reaction should be carefully balanced against simply using fresh plates considering the product itself is not free either.

The product sheet provides instruction for cleaning/treating PCR tubes hence I assume you use the same protocol for plates if you have the plate centrifuges available in your lab. You can get rid of the solutions by tapping the plates on a tissue to get the liquid out of the wells.

One of the claimed virtues of RT-PCR plates is their inability to bind DNA. (See for example the assortment at ). As such, all you need to do to remove the DNA (genomic, amplified, or primers) is to rinse them. This suggests that rinsing may also work for dNTP.

On the other, many types of RT-PCR plates are made of polycarbonate, which is able to bind protein. I would be concerned about an uneven amount of polymerase sticking to the well. To go around that, you should re-use, in any PCR plate, only those wells that saw polymerase before.

I have no idea whether other molecules bind to the wells, but presumably they fall on the DNA or protein paradigm.

It will cost you only one plates and regents for as little as four reactions to test whether these plates are reusable. In the first run, measure two samples, A and B, for some transcript. Rinse the wells, and measure samples A and B again. If the transcript ratio A/B is the wildly different between first and second attempt, you know you can't reuse them. I would also be concerned if it will take many more cycles to reach the plateau stage at the second attempt. Passing these tests, you may be all set. Failing these tests, you may still be able to use the remaining wells. Let us know.

6 Ways to Minimize Contamination during PCR

The sensitive nature of PCR allows scientists to extract and amplify useful DNA profiles. However, this high level of sensitivity can also create problems because the wrong template can be amplified. In this blog entry, we are going to explore some tips to have in mind to include in our protocol.

  • 1) Introduction
  • 2) Laboratory construction
  • 3) Unidirectional Workflow
  • 4) Pipetting Technique
  • 5) Frequently Changing Gloves
  • 6) Aseptic Cleaning Technique
  • 7) Include controls in your protocol

1. Introduction

The study of gene expression in a cell or tissue at a particular moment gives an insight into the capacity of the cell for protein synthesis. Gene expression assays, for example, gene profiling, are an important tool and are widely used in nanotoxicity studies. There are several methods available to determine gene expression, such as northern blot analysis, ribonuclease protection assay (RPA), serial analysis of gene expression (SAGE), reverse transcription-polymerase chain reaction (RT-PCR), quantitative real-time polymerase chain reaction (qRT-PCR), PCR arrays, and microarrays. Among these techniques, Northern blot analysis remains a standard method for detection and quantitation of mRNA levels despite the advent of more robust techniques. Northern blotting involves the use of electrophoresis to separate RNA samples by size, then detect the mRNA with a hybridization probe complementary to part of the target sequence. RPA is an extremely sensitive technique for the quantitation of specific RNAs in solution. It can be performed on total cellular RNA or poly(A)-selected mRNA as a target. SAGE method, as well as PCR arrays and microarrays, is used to study partial or global gene expression in cells or tissues in various experimental conditions. In this chapter, we will describe the methods to determine gene expression by using RT-PCR and real-time PCR. RT-PCR as a relatively simple, inexpensive, extremely sensitive and specific tool to determine the expression level of target genes. Real-time PCR is a quantitative method for determining copy number of PCR templates, such as DNA or cDNA, and consists of two types: probe-based and intercalator-based. Probe-based real-time PCR, also known as TaqMan PCR, requires a pair of PCR primers and an additional fluorogenic oligonucleotide probe with both a reporter fluorescent dye and a quencher dye attached. The intercalator-based (SYBR Green) method requires a double-stranded DNA dye in the PCR which binds to newly synthesized double-stranded DNA and generates fluorescence. Both methods require a special thermocycler equipped with a sensitive camera that monitors the fluorescence in each sample at frequent intervals during the PCR. The principle techniques underlying both RT-PCR and real-time PCR are total RNA isolation, reverse transcription (RT), and PCR. Reverse transcription involves the synthesis of DNA from RNA by using an RNA-dependent DNA polymerase. PCR can amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. Here we will introduce detailed procedures for RT-PCR and real-time PCR.




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PCR Plates-96 well Plates

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White Plate or Clear Plate?

Standard Profile vs. Low Profile?

Skirted Plate vs. Non-skirted Plate?

Recommended Product

See below for our PCR Plate Selection Guide, or Ask us if you need help.


White Vs. Clear:

White plates and tubes direct significantly more signal back to the qPCR instrument's detector than traditional clear plates. The improved signal reflection ensures that even the lowest levels of fluorescence are detected. White well walls also stop signal from passing through to the cycler block where it can be inconsistently reflected or absorbed, leading to increase noise levels in detected fluorescence. This keeps variations in the cycler block from affecting qPCR data. Our white PCR plastics increases sensitivity, reduces variability and increases the signal-to-noise ratio within the qPCR assay.

Clear plates provide peace of mind in being able to verify pipetting steps right after each pipetting action, as you can see the reagent added into the well as you intended. Clear plates are suitable for most applications.

These considerations also apply to 8-strip wells and individual PCR tubes.

Standard Profile vs. Low Profile

Standard-profile (full-height around 21.0 mm) wells are “standard” relative to the low-profile plates and fit in most classical thermal cyclers and real-time PCR detection systems, such as Bio-Rad's iCycler®, iQ™ series real-time PCR detection systems, and Applied Biosystems' regular PCR systems and DNA sequencers. These plates let you use large reaction volumes. These plates reduce risk-splashing caused cross-over contamination, minimizing the risk of well-to-well cross contamination.

Low-profile (around 15.5 mm) wells are the newer design that reduces the potential for condensate formation and offers advantages for light capture in fluorescence assays, low-volume reactions, and fast PCR. They have been recommended for newer instruments such as Bio-Rad's C1000 Touch™ and S1000™ thermal cyclers, CFX series real-time PCR detection systems, and Applied Biosystems' fast PCR systems. Low profile plates reduce dead space and increase PCR efficiency.

Please consult the manual of the PCR system, if necessary or ask us. Instruments with adjustable lids can usually use both standard-profile and low-profile wells. These instruments include Bio-Rad's 1000-series and DNA Engine® series thermal cyclers. Some systems require standard profile or low profile plates only.

These considerations also apply to 8-strip wells and individual PCR tubes.

Sem-skirted vs no skirted.

Semi-skirted plates are more warp-resistant because the rigidity provided by the skirt on the outer perimeter of the plate. These plates also allow for labeling or barcoding.

Non-skirted format compatible with most thermal cyclers. These plates can also be readily cut into sectional sizes as required by user, so that the entire plate is not wasted, when fewer than 96 wells are required for some experiments.

Standard PCR Protocol

Buffer: use proprietary or home-made 10x rxn mix eg: Cetus, Promega. This should contain: minimum of 1.5mM Mg2+, usually some detergent, perhaps some gelatin or BSA. Promega now supply 25mM MgCl2, to allow user-specified [Mg2+] for reaction optimisation with different combinations of primers and targets.

MAKE POOLED MASTER MIX OF REAGENTS IN ABSENCE OF DNA using DNA-free pipette, then dispense to individual tubes (using DNA-free pipette), and add DNA to individual reactions USING PLUGGED TIPS.

OVERLAY REACTIONS WITH 50UL OF HIGH-QUALITY LIQUID PARAFFIN OR MINERAL OIL to ensure no evaporation occurs: this changes reactant concentrations. NOTE: latest wisdom has it one can use VASELINE - this also allows "HOT START" PCR.

USE PLUGGED PIPETTE TIPS: prevents aerosol contamination of pipettes.

Use of detergents is recommended only for Taq from Promega (up to 0.1% v/v, Triton X-100 or Tween-20). DMSO apparently allows better denaturation of longer target sequences (>1kb) and more product.


Remember sample volume should not exceed 1/10th reaction volume, and sample DNA/NTP/primer concentrations should not be too high as otherwise all available Mg2+ is chelated out of solution and enzyme reactivity is adversely affected. Any increase in dNTPs over 200uM means [Mg2+] should be re-optimised.


Low primer, target, Taq, and nucleotide concentrations are to be favoured as these generally ensure cleaner product and lower background, perhaps at the cost of detection sensitivity.

Recommended Reaction Conditions:

Initial Conditions:

Initial denaturation at start: 92 - 97oC for 3 - 5 min. If you denature at 97oC, denature sample only add rest of mix after reaction colls to annealing temperature (prevents premature denaturation of enzyme).

Initial annealing temperature: as high as feasible for 3 min (eg: 50 - 75oC). Stringent initial conditions mean less non-specific product, especially when amplifying from eukaryotic genomic DNA.

Initial elongation temperature: 72oC for 3 - 5 min. This allows complete elongation of product on rare templates.

Temperature Cycling:

  • 92 - 94 o C for 30 - 60 sec (denature)
  • 37 - 72 o C for 30 - 60 sec (anneal)
  • 72 o C for 30 - 60 sec (elongate) (60 sec per kb target sequence length)
  • 25 - 35 cycles only (otherwise enzyme decay causes artifacts)
  • 72 o C for 5 min at end to allow complete elongation of all product DNA

"Quickie" PCR is quite feasible: eg, [94 o C 30 sec / 45 o C 30 sec / 72 o C 30 sec] x 30, for short products (200 - 500 bp).


DON'T RUN TOO MANY CYCLES: if you don't see a band with 30 cycles you probably won't after 40 rather take an aliquot from the reaction mix and re-PCR with fresh reagents. See here.

"Hot Start" PCR:

In certain circumstances one wishes to avoid mixing primers and target DNA at low temperatures in the presence of Taq polymerase: Taq pol is almost as efficient as Klenow pol at 37 o C consequently, if primers mis-anneal at low temperature prior to initial template denaturation, "non-specific" amplification may occur. This may be avoided by only adding enzyme after the initial denaturation, before the reaction cools to the chosen annealing temperature. This is most conveniently done by putting wax "gems" TM into the reaction tube after addition of everything except enzyme, then putting enzyme on top of the gem: the wax melts when the temperature reaches +/-80 o C, and the enzyme mixes with the rest of the reaction mix while the molten wax floats on top and seals the mix, taking the place of mineral oil. Information is that "gems" may be substituted by Vaseline TM .

Asymmetric PCR for ssDNA Production:

Simply use a 100:1 molar ratio of the two primers (eg: primer 1 at 0.5uM, primer 2 at 0.005uM). This allows production of mainly ssDNA of the sense of the more abundant primer, which is useful for sequencing purposes or making ssDNA probes.

Detecting Products:

Take 1/10th - 1/3rd of the reaction mix CAREFULLY from under the oil or from under the Vaseline or solidified wax, using a micropipette with plugged tip, IN AN AREA AWAY FROM YOUR PCR PREPARATION AREA!

Mix this with some gel loading buffer(1:1 - 1:5 mix:loading buffer): this is TBE containing 10 - 20% glycerol or sucrose and a dash of bromophenol blue (BPB) tracking dye.

Load 5 - 30ul of sample into wells of 0.8 - 3.0% submarine agarose gel made up in TBE, preferably containing 50ng/ml ethidium bromide.

Run at 80 -120 volts (not too slow or small products diffuse not too fast or bands smear) until BPB reaches end of gel (large products) or 2/3 down gel (small products). Use DNA markers going from 2kb down to 100 bp or less (recommend BM PCR markers).

View on UV light box at 254 - 300 nm, photo 1 - 5 sec.

Small products are best seen on 3% agarose gels that have been run fast (eg: 100 volts), with BPB run to ½ - 2/3 down the gel. It is best to include EthBr in the gel AND in the gel buffer , as post-electrophoresis staining can result in band smearing due to diffusion, and if there is no EthBr in the buffer the dye runs backwards out of the gel, and smaller bands are stripped of dye and are not visible.

NUSIEVE TM gel (FMC Corp) can also be used for small products - better resolution than agarose.

Polyacrylamide gels can be silver stained by simple protocols for extreme sensitivity of detection.

Gels can be blotted directly after soaking in 0.5M NaOH / 1.5M NaCl for 10-20 min: "dry blotting" works well (eg: gel is over- and under-layered with paper towel stacks and pressed bands transfer up and down), as does classic "Southern" blotting. Bands blotted in this way are already covalently fixed onto nylon membranes, and simply need a rinse in 5xSSPE before prehybridisation.

The example shown is of detection of Human papillomavirus type 16 (HPV-16) DNA amplified from cervical biopsy samples ( Williamson A-L, Rybicki EP (1991) Detection of genital human papillomaviruses by polymerase chain reaction amplification with degenerate nested primers. J Med Virol 33: 165-171). The left panel is a photo of an EthBR-stained 2% agarose gel the right is an autoradiograph of a Southern blot probed with 32 P-labelled HPV-16 DNA. Note how much more sensitive blotting is, and how much more specific the detection is.

Labelling PCR Products with Digoxigenin

PCR products may be very conveniently labelled with digoxigenin-11-dUTP (Boehringer-Mannheim) by incorporating the reagent to 10-35% final effective dTTP concentration in a nucleotide mix of final concentration 50-100uM dNTPs (Emanual, 1991 Nucleic Acids Res 19: 2790). This allows substitution to a known extent of probes of exactly defined length, which in turn allows exactly defined bybridisation conditions. It is also the most effective means of labelling PCR products, as it is potentially unsafe and VERY expensive to attempt to do similarly with 32P-dNTPs, and nick-translation or random primed label incorporation are unsuitable because the templates are often too small for efficient labelling.

Make a DIG-dNTP mix for PCR as follows:


  • Dig-11-dUTP 350 uM
  • dTTP 650 uM
  • dATP 1 mM
  • dCTP 1 mM
  • dGTP 1 mM

For each 50 ul of probe synthesized, a 1/10 dilution is made of the DIG-nucleotide mix when added to the other reagents as described above. The products may be analyzed by agarose gel electrophoresis - NOTE: PRODUCTS ARE LARGER THAN NON-SUBSTITUTED PRODUCT - and detected directly on blots immunologically. Probes can be used as 5-10 ul aliquots directly from PCR product mixes, mixed with hybridisation mix and denatured. Probes can be re-used up to 10 times, stored frozen in between experiments and boiled to denature.

Cleaning PCR Products

  • Getting rid of mineral oil: simply add 50ul of chloroform to the reaction vial, vortex and centrifuge briefly, and remove the "hanging droplet" of AQUEOUS solution with a micopipette.
  • Getting rid of wax or Vaseline: simply "spear" wax gem and remove do as for oil or bottom-puncture tube and blow out aqueous drop for Vaseline.
  • Cleaning-up DNA: 3 options
    • a protocol which gives DNA that is clean enough for sequencing is the following: increase volume to 100ul with water, add 10M ammonium acetate soln. to 0.2M final concentration (1/5th volume), add equal volume of isopropanol (propan-2-ol), leave on bench 5 min, centrifuge 20 min at 15 000 rpm, remove liquid using pipette, resuspend in 100ul water or TE, repeat precipitation.
    • Simply do a phenol-CHCl3 extraction (add 20ul phenol to aqueous phase, vortex, add 50ul CHCl3, vortex, centrifuge, remove UPPER aqueous phase, repeat CHCl3 extraction).
    • Make aqueous phase up to 400ul, and spin through Millipore Ultrafree-MC NMWL 30 000 cartridges (at 6000 rpm in microcentrifuge), wash through with 2x400ul water, collect +/-20ul sample: this is pure enough for sequencing.

    Product is clean enough for restriction digest immediately after reaction however, phenol-chloroform clean-up is recommended as a minimum.

    Sequencing PCR Products:

    This is best done using ssDNA generated by asymmetric PCR, and the "limiting" primer for sequencing. However, efficient sequencing of dsDNA generated by normal PCR is possible using the modification to the SequenaseTM protocol published by Bachmann et al. (1990) (NAR 18: 1309). CLEAN DNA is resuspended in sequencing buffer containing 0.5% Nonidet P-40 and 0.5% Tween-20 and sequencing primer, denatured by heating to 95oC for 5 min, snap-cooled on wet ice, and sequenced by the "close-to-primer" protocol (eg: dilute extension mixes).

    Cloning PCR Products

    T-A Cloning Strategy: Taq and other polymerases seem to have a terminal transferase activity which results in the non-templated addition of a single nucleotide to the 3'-ends of PCR products. In the presence of all 4 dNTPs, dA is preferentially added however, use of a single dNTP in a reaction mix results in (relatively inefficient) addition of that nucleotide. This complicates cloning, as the supposedly blunt-ended PCR product often is not, and blunt-ended cloning protocols often do not work or are very inefficient. This can be remedied by incubation of PCR products with T4 DNA pol or Klenow pol, which "polishes" the ends due to a 3'->5' exonuclease activity (Lui and Schwartz, 1992 BioTechniques, 20: 28-30). However, this terminal transferase activity is also the basis of a clever cloning strategy: this uses Taq pol to add a single dT to the 3'-ends of a blunt-cut cloning vector such as pUC or pBluescriptTM, and simple ligation of the PCR product into the now "sticky-ended" plasmid (Marchuk et al., 1990 NAR 19: 1156).

    Incorporation of Restriction Sites in Primers: Although this may be rendered simple by incorporating the same or different restriction sites at the 5'-ends of PCR primers - which allows generation of sticky ends and straightforward cloning into appropriate vectors - these should have AT LEAST two additional bases 5' to the recognition sequence to ensure that the enzymes will in fact recognise the sequence - and it is often found that even when this is done, the efficiency of cutting of fresh product is next to zero. This can sometimes be remedied by incubating fresh product with Proteinase K (to digest off tightly-attached Taq pol), but often is not. A solution to the problem is to use the "Klenow-Kinase-Ligase" (KKL) method: this involves "polishing" products with Klenow, kinasing them to get 5'-phosphorylation (NB: OLIGONUCLEOTIDE PRIMERS NORMALLY HAVE NO 5'-PHOSPHATES. ), ligating the fragments together to get concatemers, then restricting these with the appropriate restriction enzymes to generate the sticky-ended fragments suitable for cloning (Lorens, 1991 PCR Methods and Applications, 1: 140-141).

    Plates, Strips and Sealing for PCR, Real Time PCR & Sequencing 24 Pages

    EuroClone PCR consumables are made for a variety of thermal cyclers, real-time PCR systems and sequencers for optimal cycling performance. Manufacturing & Quality Control All plastic consumables are produced under clean-room conditions in modern injection moulding facilities. Particles, bacterial cells and other contaminants are filtered form the atmosophere. Products undergo a wide range of QC inspections during and after the production process. Visual and biological tests ensure both the absence of contaminants and the integrity of the quantitative PCR. In particular, the absence of.

    FrameStar® plates Primo FrameStar® 2 components plates Primo FrameStar® PCR plates maximise thermal stability at high temperatures preventing sample loss by minimising thermal expansion during PCR. The two-component design combines the advantages of thin wall polypropylene tubes for optimum PCR results and a rigid polycarbonate skirt and deck for highest thermal stability and rigidity. In contrast to standard onepiece PCR plates, evaporation from corner positions and outer rows is minimal, allowing for downscaling of reagent volumes and cost saving. &bull Two-component technology reduces.

    FrameStar® plates Primo® Framestar® 384 Well Designed for high-throughput PCR, FrameStar® 384 is compatible with the majority of 384 block PCR, qPCR and sequencing instruments. The rigid two-component design eliminates warping and distortion during PCR making it ideal for use with robotic systems. Cat. Primo® Framestar® 384 well plate clear wells Ideal for use with robotic systems Alphanumeric grid reference Compatible with majority of 384 block PCR, qPCR 30 µl recommended working capacity (55 µl max capacity) Plate width: Plate depth: Distance to centre of A1 from top edge: Distance to.

    FrameStar® plates Primo® Framestar® 96 Semi skirted Standard Profile (cut corner A12) Specifically designed to be directly compatible with all major thermal cyclers including all ABi instruments, this plate can be used directly in ABi 96well instruments with no adapters necessary. The rigid two-component design eliminates warping and distortion during PCR making it ideal for use with robotic systems. The semi-skirt allows for labelling or barcoding. The plate is available also with upstand (ECPCR0730C). Cat. Primo® Framestar® 96 well plate, semi-skirted, clear wells Primo® Framestar® 96.

    Standard Plates Standard Plates The thin-walled tubes of EuroClone standard PCR plates maximise heat transfer and the raised rims facilitate sealing. The range consists of non-skirted, two semi-skirted and a fully skirted plate. The non-skirted plates are available also as Tear-A-Way PCR plates, two types of plates perforated either in the vertical direction, tearing into 8well strips, or in the horizontal direction, tearing into 12well strips. Primo® 96 well Plate, Non-skirted Compatible with most thermal cyclers and sequencers Clear wells Black grid reference for easy sample.

    Standard Plates Primo® 96 well Plate, Semi-skirted Compatible with most thermal cyclers and sequencers Clear wells Black grid reference for easy sample identification Raised well rims prevent cross contamination and facilitate sealing Suitable for cap sealing (ECPCR0751 and ECPCR0752), heat and adhesive sealing 250 µl recommended working capacity (300 µl max capacity) Plate width: Plate depth: Primo 96 well plate Semi-skirted, clear wells Plate width: Plate depth: Distance to centre of A1 from top edge: Distance to centre of A1 from left edge: Pitch (distance between A1 and A2): Well.

    Standard Plates Primo® 96 well Plate, Skirted, low profile Compatible with most thermal cyclers and sequencers Clear wells, low profile Black grid reference for easy sample identification Raised well rims prevent cross contamination and facilitate sealing Suitable for cap sealing (ECPCR0751 and ECPCR0752), heat and adhesive sealing 150 µl recommended working depth: 85,48±0,50 mm Plate capacity (200 µl max capacity) Plate width: Well diameter (c): Distance to centre of A1 from top edge: Primo 96 well plate Skirted, Low Profile, clear 50 14,38±0,25 mm Distance to centre of A1 from left edge.

    PCR Tubes & Strips EuroClone tubes and cap strips are manufactured from virgin polypropylene in a Class 7 ISO certified cleanroom production facility. Strips are available in standard format and also with low profile tubes. Primo 0.2ml Individual PCR tubes, flat caps Flat and domed cap designs Primo® 0.2ml Individual PCR tubes, domed caps Suitable for all standard 0.2ml block thermal cyclers 0.25 ml recommended working capacity (0.3 ml max capacity) Suitable for most standard thermal cyclers Individually numbered tubes Available with domed or flat optical caps RNase, Dnase, human genomic.

    Primo® low profile Tube Strips Primo low profile PCR tube strips are available in clear polypropylene for standard PCR techniques. For fluorescent detection, like qPCR low profile PCR strips are available with white well tubes which give the highest sensitivity and the highest consistency as most of the fluorescence is reflected back to the detector. Cat. Description Primo Low profile 8 tubes/strip. Clear wells + flat optical caps Primo® Low profile 8 tubes/strip. White wells + flat optical caps Available with either clear or white tubes Supplied with flat optical caps RNase, Dnase, human.

    Adhesive Sealing Tapes Adhesive Sealing Tapes EuroClone offers a wide range of adhesive sealing materials processed under strictly controlled environmental conditions and certified free from DNase, RNase and human genomic DNA. Primo® PCR Seals A strong polyester transparent adhesive seal recommended for PCR but it can also be used for qPCR and other optical applications. This seal enables a high seal integrity and efficiently prevents sample evaporation. The seal can be easily peeled from the plate. The PCR seal is also available in a flexible format with perforated sheets to enable tearing.

    Parting Words

    While these tips may seem like common sense to qPCR experts, they should help newcomers to save a lot of time and money, and well as maximize their chances of getting consistent results! There are also many online support resources, including a very active Yahoo List group. Fortunately, there are many experts who enjoy helping others master the art of qPCR.

    If you want to share your best practices tips, get in touch with us by writing in the comments field!


    Originally published on January 20, 2009. Revised and updated in May 2017.

    Watch the video: What is Polymerase Chain Reaction? PCR Explained (February 2023).