Important note for all re-orderers (21.12.2019):
- Eurofins recently optimised the 2-step PCR workflow. Consequently we defined new sample preparation and ordering guidelines.
- This includes the shipment container and the required use of NGS barcodes and the adaptor sequences to be added to the target specific sequences.
- Please read the sample preparation guide carefully.
- In case you have any questions regarding those modifications or need assistance due to the changed ordering process / order sheet, please don't hesistate to contact our support team.
NGSelect Amplicon 2nd PCR
Just let us do the barcoding and pooling work for you
NGSelect Amplicon 2nd PCR starts from PCR samples prepared in your lab with universal adaptors. Eurofins Genomics purifies each PCR product and performs a 2nd PCR step, the index PCR. This PCR step introduces indexed sequencing adaptors nescessary for sample discrimination for pooled sequencing.
For sample preparation you just need to add a sequence adaptor to your target specific primers that is given in the sample submission guide. For amplification of the same target region in a series of samples, you thus need only one primer pair per target.
Highlights NGSelect Amplicons 2nd PCR:
- No need for your own barcoding - only one primer pair per target region
- No need for exact quantification or purification of amplicons
- Cost-effective outsourcing of the remaining steps
- Easy primer design and primer ordering through Eurofins
You have a large number of samples that you would like to analyse using this workflow? In these cases we can offer a customised project with full MiSeq runs. Please contact us for a project discussion.
Your amplicon size is different to what we request here? Please also have a look at the NGSelect Adaptor Ligation workflow.
You Need Oligos to prepare your Amplicons? Click here
|Illumina 2nd PCR library
25 µl of unpurified amplicon (PCR product)*
Amplicon size (not including the universal adaptors) between 340 and 760 bp; for full-length sequencing smaller than 570 bp
Quality check need to be performed by agarose gel electrophoresis (= clear visible band(s) of expected size using 5 µl PCR product).
Samples need to be shipped in 96-well PCR plates with 200 µl well volume.
- approx. 80 - 90 k read pairs per amplicon
- 2 x 300 bp read mode
15 - 20 working days for up to 96 samples.
* For less starting material please contact us.
How to prepare your Amplicons for NGSelect Amplicons 2nd PCR product
It's as simple as can be
- Define the loci(s) you want to amplify and design target specific primer sequences that amplify a region of 340 - 760 bp. For full length sequencing please plan with a max. amplicon length of 570 bp.
- Upload your target specific sequence on our corresponding oligo order page and let Eurofins do the automatic and validated primer design.
- Oder NGSgrade primers and prepare amplicons with the designed primers.
- Check amplification success on a gel and send unpurified amplicons to Eurofins.
Any questions or support required? Please contact us.