Dilute primers

• Lyophilized primers 
  
  Add the following amount of nuclease-free water to the lyophilized primers in order to obtain a 100x stock solution :

​

• 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

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 :

Separate delivery of forward and reverse primers :

* 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

Perform the PCR cycling program

* 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.

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).