Frequently asked questions
What does the assay quality label (, or stars) mean?
While we did our very best to design perfect PCR assays, we are sometimes confronted with the constraints of the human genome sequence. As such, we occasionally needed to make a compromise (i.e. allowing a SNP in the primer annealing region or acknowledging possible off-target amplification). 73.6% of our assays are labeled as perfect () meaning that we predict very high or ultimate specificity (because of the presence of at least 3 mismatches in each primer relative to a potential off-target sequence) and the complete absence of SNPs with a minor allele frequency higher than 1%. 10.9% of the assays are labeled as good () meaning that we predict good specificity (at least 2 mismatches per primer) and a maximum of 1 SNP. 15.5% of the assays are labeled as fine (). In this category, we had to make compromises. Each assay comes with detailed information on predicted specificity and presence of possible SNPs. There is a very good correspondence between the predicted assay quality and the sequencing results (see product brochure, our peer-reviewed paper (Coppieters et al., 2016) or here). Assays marked with orange stars () have been wet-lab validated, while assays labeled with blue stars () have only been in silico evaluated.
There is a very good correspondence between the predicted assay quality and the sequencing results (see product brochure). If the specificity info - reachable by clicking on the stars assocated with a certain assay - in the assay detail windows shows that there are hits with 2/3 mismatches, this means that 2 and 3 mismatches are present in the forward and reverse primer, respectively.
Can I add universal tails to my primers?
Yes, you can. Universal tails can be added in two ways. One way is by supplying forward and reverse universal tail sequences in you profile and selecting "Yes" in the "Automatically add universal tail sequences?" field. This will automatically add the corresponding universal tails when adding assays to your cart. In addition, you have the option to add custom tail sequences to (a selection of) your primers once assays have been added to your cart. To do this, go to your cart and first select the assays for which you wish to add tail sequences. Next, click on the 'add/delete tails' submenu item in the 'action'-button menu and enter your forward and/or reverse tail sequence in the dialog box. In the same way, you can remove tail sequences from your assays by selecting the desired assays and entering no sequences in the dialog box. Adding tails comes at no extra cost.
Amplification guidelines
These are the universal PCR amplification guidelines for use with the pxlence PCR assays.
1.
Dilute primers
Lyophilized primers :
Add the following amount of nuclease-free water to the lyophilized primers in order to obtain a 100x stock solution :
N° of reactions
µl water to be added
100
20
500
100
1000
200
Primers resuspended in buffer are delivered in a 100x stock solution (10 mM Tris, pH 8.0, 0.1 mM EDTA).
100x stock primers should be stored in a (frost free) freezer at -20 °C.
Perform the following dilution to make 100 µl of a 10x working solution :
10 µl 100x stock primer + 90 µl nuclease-free water
2.
Make PCR mix
We recommend to use the following PCR kit :
KAPA2G Robust HotStart ReadyMix (KAPABiosystems, KK5701, KK5702).
This kit contains all necessary components to perform a PCR reaction; only primers and template DNA need to be added.
Make the following PCR reaction mix (total volume of 20 µl) :
Pre-mixed delivery of forward and reverse primers :
Reagent
µl per reaction
KAPA2G Robust ReadyMix
10
Mix of forward and reverse primer (10x)
2
Template DNA (5-50 ng)
2 to 8 *
Nuclease-free water
6 to 0 **
Separate delivery of forward and reverse primers :
Reagent
µl per reaction
KAPA2G Robust ReadyMix
10
Forward primer (10x)
2
Reverse primer (10x)
2
Template DNA (5-50 ng)
2 to 6 *
Nuclease-free water
4 to 0 **
* we don't recommend to pipet less than 2 µl template DNA; please dilute DNA to add at least 2 µl.
** add water up to 20 µl total volume
3.
Perform the PCR cycling program
 
Duration
Temperature
 
Activation
3 min
95 °C
 
Denaturation
Annealing
Elongation
15 sec
10 sec
15 sec
95 °C
60 °C
72 °C
 
35-40 cycles *
 
Final elongation
1 min
72 °C
 
* 35 cycles are sufficient if 50 ng of DNA is used; when using 5 ng, we recommend to increase the number of cycles to 40.
4.
Downstream applications
Both Sanger sequencing and next-generation sequencing (NEBNext® DNA Library Prep, New England BioLabs; Nextera® XT DNA Library Prep, Illumina) have been performed successfully on PCR products generated with the pxlence assays. Please contact us if you need further advice on these downstream applications ().
How do I order pxlence PCR assays?
We only sell through our online shop. In short, search for the assays of interest, add them to your cart and checkout. We accept bank/wire transfer payments. All payments are in Euro currency. You can also purchase credits, and use the credit for future orders.
Can pxlence assays be used in a multiplex PCR?
Our assays are carefully designed to have uniform primer annealing temperatures and amplicon lengths, to facilitate multiplexing. We have successfully multiplexed several assay panels for simultaneous equimolar amplification of DNA targets. For multiplex use, we recommend a dedicated multiplex PCR mastermix and reduced primer concentrations (20 nM final concentration for a given assay is typically a good starting point; to achieve uniform end-point amplicon concentrations, assay specific optimization of primer concentrations may be required.
Do you offer customized PCR assay design?
Yes, we do. Just enter your target(s) of interest and desired amplicon size on the Custom assays page. If you click on the Add to cart button, the design will immediately be initiated upon checkout. Once completed, the assay(s) will be synthesized and delivered to you. You can also request a quote first - e.g. for larger sets of targets - by clicking on the Request quote button or by using our Contact form. If additional information is required regarding your custom design request, we will contact you.
What is the delivery time?
Every pxlence assay is made to order and undergoes extensive quality control prior to shipment. Typically, assays in tubes are shipped within four business days of ordering. Lyophilized assays in plates are shipped within five business days of ordering.
Best-in-class PCR assays, what does that mean?
We have done our utmost best to design primers that adhere to strict in silico quality controls ensuring robust and specific amplification of the target region of interest. Specifically, we avoid single nucleotide polymorphisms (SNPs) in the primer annealing regions (known to hamper efficient annealing or create allelic dropout), avoid secondary DNA structures (known to reduce PCR efficiency), and use a very stringent specificity prediction tool. Together, this ensures that the pxlence assays will amplify target DNA with very high uniformity and specificity. Several thousands of assays have been tested in the pxlence laboratory or in the lab of happy customers. Approximately 95% of the assays generate less than 2% off-target reads. More information is available in our product brochure or here.
What reaction format is available?
The pxlence assays either come as lyophilized (100, 500 or 1000 reactions) or resuspended in buffer (100, 500 or 1000 reactions). For convenient use, forward and reverse primer come premixed, but can also be ordered separately. Primers are available in tubes or 96-well deep-well V-bottom plates. There is no price difference between tubes and plates or between lyophilized and resuspended in buffer. Please see our amplification guidelines for use of pxlence assays to amplify target of interest.
Do we get access to the primer sequences?
No, this is proprietary information, but you do get the amplicon context sequence as defined in Bustin et al., Clinical Chemistry, 2013 (Primer Sequence Disclosure: A Clarification of the MIQE Guidelines ), this is the true amplicon sequence +/- 20 nucleotides. Further, we provide detailed in silico quality control parameters for all assays (presence of SNPs and specificity score), see 'What does the assay quality label (, or stars) mean?'.
What PCR conditions should I use when amplifying my target with a pxlence assay?
We recommend the Kapa2G Robust PCR kits for highly successful amplification of the target region. Several thousands of assays have been tested internally or by some of our early-access customers with very high success rate. We use the HotStart ReadyMix (kit code KK5701 or -02). pxlence assays should be used at 1x final concentration, and work with one universal PCR cycling program. Please also see our amplification guidelines.
Do I really need to confirm my massively parallel sequencing (NGS) variants?
Based on our survey with almost 200 respondents from all over the world, 70% indicated the need to confirm their exome, whole genome or targeted resequencing studies; 56% of these use Sanger sequencing, 19% massively parallel targeted resequencing and 20% both Sanger and massively parallel sequencing.
Do pxlence assays come with a diagnostic guarantee?
Pxlence assays can be used for research purpose only, data interpretation and clinical outcome remains the responsibility of the customer. There may be other licenses to use PCR in a commercial environment. It is up to the customer to ensure he/she has the required licenses.