Sample requirements for Illumina mate-pair libraries

Sample requirements for Illumina mate-pair libraries
• The DNA must be of high molecular weight (greater than 20 Kb fragments).
• Samples must be submitted free from any contaminants. Purify samples by ethanol precipitation and resuspension,
or using Agencourt Genfind v2 (Beckman). Remove all traces of ethanol after purification, as enzymatic steps in the
library preparations are inhibited by this.
• Purification should be confirmed by values of of ≥1.80 for both of the NanoDrop A260/230 and A260/280 ratios. If the
samples need further purification after submission, the additional expenses will be added to the formal quote.
• Ensure that the DNA is fully resuspended in nuclease-free water or low concentration TRIS buffer (no EDTA). If
samples and ladders of high molecular weight are not fully resuspended, they will not run properly on a gel.
• Accurate quantification of the nucleic acids in the sample(s) is necessary. Use a dye based method such as Qubit
(Invitrogen). If this is not available, use an agarose gel with a quantitative ladder.
• Please provide a gel image of all samples to confirm sample integrity, including type of ladder and/or indication of
fragment size(s). If there is more than one band or a smear, the DNA may be degraded or have a contaminant that
could affect the library preparation. RNA usually appears as a smear <200 bp. If RNA is present, please treat the
DNA with RNase A (DNase-free) and then purify, preferably using a column based method.
Gel image below illustrates intact (panel A), degraded (panels B and D) and RNA contaminated (panels C and D)
genomic DNA run along Hyperladder I (Bioline). If a smear is in range between zero and ~2000 bp, only treatment
by RNase will reveal whether the degraded material is gDNA or RNA.
• The DNA should be stored at 4°C. Do not freeze the sample, as this could cause fragmentation of the DNA.
• Please supply a minimum of 4 µg gDNA in a volume of 100 ul for mate-pair libraries ranging from 3 Kb to 12 Kb.