Sample preparation:
If it has been established that a restriction enzyme (e.g. ApeKI) and method are suitable for the species you 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). To ascertain the chemical purity of the samples, the UV absorption ratios should be 1.8 to 2.0 (260/280 nm) and > 2.0 for 260/230 nm . A volume of 20 ul per sample is sufficient.
The DNA samples have to 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.
The DNA samples have to be RNA-free. Thus, the DNA isolation protocol has to include an RNAse digestion step.
Before shipping us samples please email us gel images of representative samples. RR-Seq Sequencing is carried out with single-end 100 bp or single-end 150 bp reads.
We offer dual enzyme RR-Seq library sequencing with PstI or SbfI on one end combined with MspI for the other end.
Alternatively, we do offer a restriction-enzyme-free RR-seq protocol which is PCR-based.If there are no sequencing data for these enzymes for your species of interest yet, we would need to establish that the enzymes are suitable to avoid targeting sites present in abundant repeat sequences. We will do this by carrying out test-library preparations (see below).
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