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Section 3.2 Prepare a 2 × 25 bp mate-paired library
Shear the DNA
1. Add 4 volumes of Binding Buffer (B2-S) with isopropanol (55%) to 1 volume of
sample. Mix well.
2. Apply about 700 μL of the sample in the binding buffer to the PureLink™
column(s) in collection tube(s).
3. Centrifuge the column(s) at 10,000 × g for 1 minute, then discard the flowthrough.
4. Repeat steps 2 and 3 until the entire sample has been loaded onto the column(s).
Place the column(s) back into the same collection tube(s).
5. Add 650 μL of Wash Buffer (W1) with ethanol to wash the column(s).
6. Centrifuge the column(s) at 10,000 × g for 1 minute, then discard the flowthrough. Repeat centrifugation at maximum speed to remove residual wash buffer.
7. Transfer the column(s) to clean 1.5-mL LoBind tube(s).
8. Add 50 μL of Elution Buffer (E1) to the center of the column(s) to elute the DNA,
then let the column(s) stand for 1 minute at room temperature.
9. Centrifuge the column(s) at maximum speed for 1 minute.
10. Add the eluate from step 9 back to the column(s), then let the column(s) stand for
1 minute at room temperature.
11. Centrifuge the column(s) at maximum speed for 1 minute.
12. If necessary, pool the eluted DNA.
13. Quantitate the purified DNA by using 2 μL of the sample on the NanoDrop® ND1000 Spectrophotometer (see “Quantitate the DNA with the NanoDrop® ND-1000
Spectrophotometer” on page 189).
Store the purified DNA in Elution Buffer (E1) at 4 ºC, or proceed
directly to “End-repair the sheared DNA” on page 98.
STOPPING POINT.
SOLiD™ 4 System Library Preparation Guide
97
Chapter 3 Mate-Paired Library
Preparation
Purify the DNA with
the SOLiD™ Library
Column Purification
Kit
3