58 Library Preparation

1. Prepare DNA in nuclease-free water by transferring ~400 ng of genomic DNA into a DNA LoBind tube and adjust the volume to 7.5 µl with nuclease-free water.

a. Flick the tube to avoid shearing.

b. Spin down briefly using a microfuge.

2. In a 0.2 ml PCR tube, mix:

a. 7.5 µl of 400 ng genomic DNA

b. 2.5 µl of Fragmentation Mix RB01-12 (one “number” or “barcode” for each sample)

c. The total volume is 10 µl. Mix gently by flicking the tube and spin down.

3. Incubate the tube at 30°C for 1 minute and then 80°C for one minute in a thermocycler.

a. Put the tube on ice to briefly cool down.

4. Now, pool the barcoded samples in a 1.5 ml Eppendorf DNA LoBind tube and expect to have ~10 µl per sample.

a. Note: if barcoding more than four samples, increased yield can be achieved by using AMPure XP beads for a cleanup of all barcoded samples. This is an optional step.

5. Add 1 ul of RAP to 10 µl of barcoded DNA.

6. Incubate at room temperature for five minutes. Then place on ice.

7. At this point, prime the flow cell (as noted above).

8. In a new tube, prepare the library by adding:

a. 34 µl of Sequencing Buffer (SQB)

b. 25.5 µl of Loading Beads (LB), mixed immediately before use

c. 4.5 µl of nuclease-free water

d. 11.0 µl of DNA library

e. The total volume is 75 µl.

f. Load the library onto the flow cell immediately after adding the SQB and LB because the fuel in the buffer will start to be consumed by the adapter.

9. Cover the flow cell to protect from light with the black plastic films. Wait ten mintues for the sample to enter the pores.

10. Start MinKNOW software by setting to Sequence and identifying the kit and output directory.