We currently offer restriction-enzyme based reduced representation sequencing with ApeKI, MspI, NlaIII, PstI and SbfI enzymes, as well as a strictly PCR-based reduced representation sequencing protocol.
Sample preparation: If it has been established that enzyme (e.g. ApeKI) and method are suitable for the species your are working on (please see below), we require the samples for RR-Seq to be submitted in a 96-well plate. One or two wells should remain empty for negative controls. The concentration of the samples should be normalized to 50 ng/ul as assayed by an intercalating dye (fluorometry using a Qubit; Quantus, or plate reader). The UV absorption ratios should be 260/280 nm 1.8 to 2.0 and 260/230 > 2.0 . A volume of 20 ul per sample is sufficient. We understand that the DNA amounts might be difficult to achieve for smaller organisms. We will try to work around such limitations, but can't promise perfection in these cases.
For the restriction-enzyme-based protocols the DNA samples should be extracted using a CTAB-free protocol (best a spin column protocol), since very precise DNA sample quantification is critical for the success of the protocol and CTAB interferes with precise quantifications. The DNA samples also have to be RNA-free. Thus, the DNA isolation protocol has to include an RNAse digestion step. The DNA has to be dissolved in EB-buffer or TLE-buffer (Tris-Low-EDTA-buffer). Do not use TE-Buffer or AE-buffer.
For restriction-enzyme based reduced representation sequencing, the samples should be normalized to 50 ng/ul as assayed by an intercalating dye (fluorometry using a Qubit; Quantus, or plate reader).
For the PCR-based based reduced representation sequencing the samples should be normalized to 10 ng/ul or 20 ng/ul as assayed by an intercalating dye (fluorometry using a Qubit; Quantus, or plate reader).
We understand that the DNA amounts might be difficult to achieve for smaller organisms. We will try to work around such limitations, but can't promise perfection in these cases.
Before shipping us samples please email us gel images of representative samples. RR-Seq Sequencing is carried out with paired-end reads on AVITI or NovaSeq X sequencers.