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Section 3.1 Prepare a 2 × 50 bp mate-paired library
Shear the DNA
1. In a round-bottomed 13-mm × 65-mm borosilicate tube, dilute 10 to 20 μg of
DNA in 500 μL solution so that the final volume contains 20% glycerol in
Nuclease-free Water (see Table 13).
Chapter 3 Mate-Paired Library
Preparation
Shear the DNA with
the Covaris™ S2
System
3
Note: To prepare a short-insert (≤1 kb) mate-paired library from a small
genome, ≥5 μg of DNA is sufficient.
Table 13
Dilute the DNA for shearing
Component
Amount
UltraPure™ Glycerol
100 μL
DNA
10 to 20 μg
Nuclease-free Water
Variable
Total
500 μL
2. Shear the DNA using the Covaris™ S2 System shearing program described in
Table 12 on page 54.
3. Transfer 500 μL of sheared DNA into a clean 1.5-mL LoBind tube.
4. Wash the borosilicate tube with 100 μL of Nuclease-free Water and transfer the
wash to the 1.5-mL LoBind tube. Mix by vortexing and then proceed to “Purify
the DNA with the SOLiD™ Library Column Purification Kit” on page 56.
Shear the DNA with
the HydroShear®
DNA Shearing
Device
1. In 1.5-mL LoBind Tubes, dilute 10 to 20 μg of DNA to 150 μL with Nuclease-free
Water. If you are starting with an input >20 μg, split the DNA into ≤20 μg aliquots
and shear each aliquot in 150-μL volume. For better coverage of large and
complex genomes, more DNA should be used if it is available.
2. On the Edit Wash Scheme tab, specify the solution and cycles:
• 2 cycles of WS1 (0.2 N HCl)
• 2 cycles of WS2 (0.2 N NaOH)
• 3 cycles of Nuclease-free Water
3. Run the wash scheme on the HydroShear DNA Shearing Device.
4. Adjust the speed code (SC) and number of cycles according to Table 12 on
page 54, and adjust the volume setting to 150 μL.
5. Begin shearing. Repeat the shearing for the other aliquot of DNA, if applicable. It
is not necessary to run the wash cycle if both tubes contain the same DNA.
6. Run the wash scheme after DNA shearing is complete.
7. Pool the aliquots of sheared DNA, if applicable.
SOLiD™ 4 System Library Preparation Guide
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