8. Clean up with AMPure beads.
• 50μl after PCR.
• + 90μl AMPure XP magnetic beads.
• Gently pipet up and down 10Â.
• 5 min at room temperature.
• Place tubes on a magnet holder for 2 min (or until the liquid is clear).
• Discard supernatant.
• 2 Â 500μl of 70% ethanol for 30 s, wash magnetic beads, discard supernatant.
• Air dry the beads on magnetic stand at RT for 2–5 min or at 37
C in a heatblock.
• Elute with 30μl H 2 O, pipette the entire volume up and down 10 times to mix
thoroughly.
• 2 min at room temperature.
• ↺ 13,000 rpm, 1 s.
• Place tubes on a magnet holder for 2 min (or until the liquid is clear).
• Transfer 30μl in new tube for Blue Pippin.
9. BLUE PIPPIN (size selection of ChIP Seq Library)
Purify and Size Select the Ligation Products
• Perform the size selection using the Sage BLUE PIPPIN.
• Bring Pippin Prep loading solution to RT.
Prepare DNA Samples for loading
• Initiate the BluePippin instrumentation allowing software to launch.
• Add 10μl of RT loading solution to each sample for a total of 40μl. Mix well and
centrifuge briefly.
Program Protocol
• From main screen select “Protocol” to go to the Protocol Editor Screen. Press “New” to
open a new protocol.
• Select the appropriate cassette type.
• Select a reference lane which will contain the DNA marker.
• NOTE: Verify lane assignment of software display vs cassette lanes.
• Enter - bp target size 275 bp.
• -bp start size 250 bp. NOTE: Cut size may need to be adjusted to target optimal size
distribution of fragmented sample.
• -bp end size 300 bp.
• Enter sample name into the “Sample ID” field.
• Select “Save as” and name protocol to retain for future applications (TruSeq ChIP 250–
300 bp).
• Return to Main Screen.
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Appendix
• 50μl after PCR.
• + 90μl AMPure XP magnetic beads.
• Gently pipet up and down 10Â.
• 5 min at room temperature.
• Place tubes on a magnet holder for 2 min (or until the liquid is clear).
• Discard supernatant.
• 2 Â 500μl of 70% ethanol for 30 s, wash magnetic beads, discard supernatant.
• Air dry the beads on magnetic stand at RT for 2–5 min or at 37
C in a heatblock.
• Elute with 30μl H 2 O, pipette the entire volume up and down 10 times to mix
thoroughly.
• 2 min at room temperature.
• ↺ 13,000 rpm, 1 s.
• Place tubes on a magnet holder for 2 min (or until the liquid is clear).
• Transfer 30μl in new tube for Blue Pippin.
9. BLUE PIPPIN (size selection of ChIP Seq Library)
Purify and Size Select the Ligation Products
• Perform the size selection using the Sage BLUE PIPPIN.
• Bring Pippin Prep loading solution to RT.
Prepare DNA Samples for loading
• Initiate the BluePippin instrumentation allowing software to launch.
• Add 10μl of RT loading solution to each sample for a total of 40μl. Mix well and
centrifuge briefly.
Program Protocol
• From main screen select “Protocol” to go to the Protocol Editor Screen. Press “New” to
open a new protocol.
• Select the appropriate cassette type.
• Select a reference lane which will contain the DNA marker.
• NOTE: Verify lane assignment of software display vs cassette lanes.
• Enter - bp target size 275 bp.
• -bp start size 250 bp. NOTE: Cut size may need to be adjusted to target optimal size
distribution of fragmented sample.
• -bp end size 300 bp.
• Enter sample name into the “Sample ID” field.
• Select “Save as” and name protocol to retain for future applications (TruSeq ChIP 250–
300 bp).
• Return to Main Screen.
204
Appendix