FAQ: How can pre-shear spike-in controls be added to cfDNA or other pre-fragmented DNA (amplicons, restriction digested DNA, etc.) going into EM-seq™ workflow?
10 µl of Control DNA Unmethylated Lambda (2ng/µl)
10 µl of Control DNA CpG methylated pUC19 (0.1ng/µl)
30 µl of 15 mM Tris-HCl pH 8.0
Shear to an average size of 350 bp
The Control DNA (combined Control DNA CpG methylated pUC19 (0.1ng/µl) and Control DNA Unmethylated Lambda (2ng/µl)) sheared in a final volume of 50 µl is equivalent to a 1:5 dilution. Therefore, to further dilute these pre-fragmented controls, for example to a 1:10 dilution, a further 1:2 dilution is required.
Note: Unmethylated Lambda and CpG methylated pUC19 control DNAs are provided in 1 mM Tris pH 7.5 and 0.1 mM EDTA-containing buffer. It is critical to have at least 10 mM Tris when fragmenting these controls using mechanical shearing methods like Covaris. Using a buffer with lower than 10 mM Tris during shearing results in altered conversion metrics, and the conversion efficiencies assessed might not be accurate.
