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).

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 M13 tags to my primers?
Once assays have been added to your cart, you have the option to add custom tail sequences to (a selection of) your primers. 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' menu item 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. You can also download this document here.
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
Resuspended primers 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 **
* 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
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 15 sec 95 °C 35-40 cycles *
Annealing 10 sec 60 °C
Elongation 15 sec 72 °C
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 (support@pxlence.com).
How do I order assays?
1. Selecting and ordering assays can be done on the order page. Select the type of query you want to perform. When querying based on a gene symbol (eg. CEP290), Ensembl or RefSeq accession numbers (eg ENSG00000198707, ENST00000552810, NM_025114), chromosomal location (eg 12:88049014-88142216) or dbSNP rs-number, the assay type (long or short amplicons) need to be selected. If you perform a query using a pxlence catalogue number, no assay type selection is needed. It is also possible to perform a query using a batch file. Allowed batch file formats are a .vcf file or a list containing symbols as specified by the query string.
2. When one or more of your search strings result in multiple hits when querying our database, these will be presented to you. You will be allowed to select the identifier of your interest, aided by links referring to the external Ensembl website where more information on each hit can be found.
3. After all required information has been supplied, our catalogue will be queried for assays overlapping with each of your search string. Results will be presented in a table format as shown below, where you can customize the format of your product (resuspended/lyophilized, mixed/separate forward & reverse primers, plate/tube and number of reactions). A quality level has been assigned to each assay, more information on this can be found here.
4. The desired reaction number, item amount, format, form and primer constitution can be customized for each assay separately. However, selecting multiple assays and clicking the 'perform action on selection' button above the assay table, will display a popup dialog enabling you to adjust these values for all selected assays at once. Adding assays to your cart can be done by selecting the ones you are interested in and clicking on the 'add selected to cart' button.
5. Additional detailed assay information can be found by clicking on the quality level icon of the corresponding assay. This will open up a popup window showing the possible presence of single nucleotide polymorphisms (SNP) in the primer annealing sites and more detailed specificity information in addition to the general assay info. A more detailed explanation of the specificity information can be found here.
6. By clicking on the 'genome view' link above an assay table, a popup displaying a genomic view of the corresponding region (specified by the supplied gene symbol or chromosomal location) will open up. This popup also allows you to save this view in a .pdf format by means of the 'save image' button, while the 'go to the UCSC Genome Browser' will take you to the UCSC Genome Browser giving you more customization options. The '.bed file' link allows you to download the assay context coordinates in a .bed file format.
7. Your cart is accessible through the 'My cart' link in the upper left corner of the website. This link will take you to a webpage depicting all the products added to your cart.
8. Here, it is possible to remove items from your cart by selecting items and clicking the 'delete items' button, and to add purchase orders, quote numbers or discount codes by means of the 'add quote/PO number' button. Tag sequences (e.g. M13) can be added or removed to/from specific assays of interest by selecting them and clicking on the 'add/delete tags'. In addition, it is possible to save your cart for future use. Stored carts can be retrieved under the 'ordering history' tab in your account.
9. When going to the checkout, you will be asked to register if you don't have an account yet (through the 'I have no account' button) or to login and review you contact information in case you already have one.
10. After registration and reviewing your contact details, you will be asked to choose a payment method. Payment is possible by means of PayPal, credit card, bank transfer or pxl-credits. When choosing PayPal or credit card, you will be redirected to the PayPal website for payment processing. On the PayPal website, you have the possibility to pay by means of your PayPal account (through the 'pay with my PayPal account' link) or credit card (through the 'don't have a PayPal account' link). Only if you already purchased pxl-credits, this payment method will be displayed. Credits can be purchased under the 'pxl-credits' in your account. After payment, you will be redirected to the pxlence website and receive a purchase confirmation email. The details of your purchase can be viewed under the 'ordering history' tab in your account.
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. Please have a look at our detailed step-by-step instructions for searching and ordering the assays. We accept bank/wire transfer, credit card or PayPal payments. All payments are in Euro currency. You can also purchase credits, and use the credit for future orders.
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.
What reaction format is available?
The pxlence assays either come as lyophilized (100, 500 or 1000 reactions) or resuspended (100x, also 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. 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.