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Place the tube with its lid open in a 37°C heat block until the bead pellet gets a cracked appearance (1–2 min). Avoid overdrying. Resuspend the bead pellet with 20 ml of TE (or water). Place on the magnet for 3 min and elute into a fresh tube. The eluate contains the sample. Add another 20 ml of TE and resuspend the beads. Place on the magnet for 3 min, elute, and add to the first eluate. The sample is ready for quantification and sequencing. 9. Assessing Sample Concentration and Sequencing Sample concentration is assessed using the HeliScope™ OptiHyb assay.
The reagents have been optimized to produce correct tail lengths and stabilized to permit longterm storage. Smaller quantities of DNA can be sheared. 5 pmoles of DNA (see Note 13), as little as 100 ng of high quality DNA can be used at this step. If you notice you are removing beads during aspiration, do not attempt to remove all the beads with a p1000 pipette. Rather, remove the last 20–50 mL with a p200 pipette. If 100 ng of DNA has been sheared, the elution volumes should be reduced to 10 mL in both steps 12 and 15.
1 mL terminal transferase enzyme (20 U/mL) per sample. 5 pmole DNA tube). The mixing step is crucial to the success of the tailing reaction. 4 pmoles of DNA. The DNA should be in a final volume of 12 mL and 1 mL of a 1:2 dilution of the HelicosTM PolyA Tailing Control Oligo TR in water should be added. Two microliter of TR oligo should be used in the Oligo TR Control. 1 mL terminal transferase enzyme (20 U/mL) per sample. Seven microliters of control master mix should be added to each tube. 6).