Isolation Purification and Separation

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Authors: Radoslaw Kamil Ejsmont 

Introduction

This is a protocol for amplification of pFlyFos clones.

Procedure

  1. Inoculate 50 ml of LB+Cm25 with a single colony of FlyFos strain. Culture overnight at 37°C with vigorous shaking.
    • NOTE: Please follow ▲ or ● symbols throughout the protocol for options tailored to different volumes.
  2. Use ▲ 2×1 ml or ● 2×5 ml to inoculate ▲ 2×100 ml or ● 2×500 ml LB+Cm25+Ara0.1% in ▲ 500 ml or ● 2500 ml flasks. Culture overnight at 37°C. Shake cultures vigorously – minimum 250 rpm.
  3. Harvest the bacterial cells by centrifugation at 6000 x g for 15 min at 4°C.
  4. Resuspend the bacterial pellet from both flasks combined in ▲ 8 ml or ● 50 ml of Buffer P1.
  5. Add ▲ 8 ml or ● 50 ml of Buffer P2, mix thoroughly by vigorously inverting 4–6 times, and incubate at room temperature for 5 min.
  6. Add ▲ 8 ml or ● 50 ml of chilled Buffer P3, mix immediately and thoroughly by vigorously inverting 4–6 times, and incubate on ice for 30 min.
  7. Centrifuge at ≥20,000 x g for 30 min at 4°C. Remove supernatant containing plasmid DNA promptly.
  8. Place folded Whatmann filter in a 50 ml syringe. Prewet and compress filter by passing water through the syringe. Use such prepared syringe for filtering supernatant.
  9. Precipitate the DNA by adding ▲ 17 ml or ● 105 ml (0.7 volumes) of room temperature isopropanol to the lysate. Centrifuge at ≥15,000 x g for 30 min at 4°C, and carefully decant the supernatant.
  10. Redissolve the DNA pellet in 500 μl warm (60°C) TE buffer, pH 8.0, and add Buffer QBT to obtain a final volume of ▲ 5 ml or ● 12 ml for selected ▲ QIAGEN-tip 100 or ● QIAGEN-tip 500, respectively.
  11. Equilibrate a ▲ QIAGEN-tip 100 or ● QIAGEN-tip 500 by applying ▲ 4 ml or ● 10 ml Buffer QBT, and allow the column to empty by gravity flow.
  12. Apply the DNA solution from step 10 to the QIAGEN-tip and allow it to enter the resin by gravity flow.
  13. Wash the QIAGEN-tip with ▲ 2×10 ml or ● 2×30 ml Buffer QC.
  14. Elute DNA with ▲ 5 ml or ● 15 ml Buffer QF.
  15. Precipitate DNA by adding ▲ 3.5 ml or ● 10.5 ml (0.7 volumes) of room temperature isopropanol to the eluted DNA. Mix and centrifuge immediately at ≥15,000 x g for 30 min at 4°C. Carefully decant the supernatant.
  16. Wash DNA pellet with ▲ 2 ml or ● 5 ml room-temperature 70% ethanol, and centrifuge at ≥15,000 x g for 10 min. Carefully decant the supernatant without disturbing the pellet.
  17. Wash DNA pellet again with ▲ 2 ml or ● 5 ml room-temperature 70% ethanol, and centrifuge at ≥15,000 x g for 10 min. Carefully decant the supernatant without disturbing the pellet.
  18. Air-dry the pellet for 5–10 min, and redissolve the DNA in a suitable volume (▲ 50 μl or ● 250 μl) of warm (60°C) nulcease-free water.
  19. You should obtain in total ▲ 100 μg or ● 500 μg of pure injection-quality fosmid DNA.

Associated Publications

A toolkit for high-throughput, cross-species gene engineering in Drosophila, Radoslaw K Ejsmont, Mihail Sarov, Sylke Winkler, Kamil A Lipinski, and Pavel Tomancak, Nature Methods 6 (6) 435 - 437 24/05/2009 doi:10.1038/nmeth.1334

Author information

Radoslaw Kamil Ejsmont, MPI-CBG

Correspondence to: Radoslaw Kamil Ejsmont ([email protected])

Source: Protocol Exchange (2009) doi:10.1038/nprot.2009.109. Originally published online 27 May 2009.

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