Materials

• 1 ml of rabbit blood
• QIAamp DNA blood Midi kit
• TE buffer (1 mM EDTA, pH 8.0)
• 15 ml Falcon tubes
• Centrifuge capable of taking 15 ml Falcon tubes
• 96-100% ethanol

Method

1. Lyse the cells using the reagents and volumes according to the Qiagen QIAamp DNA blood Midi kit protocol for volumes of blood between 1–2 ml (page 19, steps 1–3).
2. Critical step: We have found that lysis for 1 hour at 60°C is more effective that the recommended 10 minute incubation at 70°C (step 4).
3. Purify the lysate according to the protocol (page 20-21, steps 5-11).
4. Critical step: To maximize the DNA yield, we recommend adding 100 µl of TE (1 mM EDTA, pH 8.0) to the membrane followed by incubation at 37°C for 10 minutes. Spin the column at 8000 rpm for 2 minutes, and collect the eluted DNA. Repeat the above step twice to give a total elution volume of 300 µl.
5. Take 60 µl of eluate (corresponding to 3 µg of DNA) and perform a SPRI size selection.

Results

• Yield: 12-16 μg (before SPRI size selection)
• OD 260/280: 1.96 (after SPRI size selection)
• OD 260/230: 2.33 (after SPRI size selection)

• Fragment size (FEMTO pulse):

Sequencing performance

Libraries were prepared using the Ligation Sequencing Kit and Rapid Sequencing Kit.

• Read length profile:

Change log

Version	Change
v1, 27th July 2019	Initial protocol publication
v2, 14th August 2023	Updated protocol page references