This is a protocol for amplification of pFlyFos clones.
Method Article
A protocol for FlyFos clone amplification
https://doi.org/10.1038/nprot.2009.109
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pFlyFos
FlyFos clones
fosmid
DNA isolation
This is a protocol for amplification of pFlyFos clones.
NOTE: Please follow ▲ or ● symbols throughout the protocol for options tailored to different volumes.
Use ▲ 2 x 1 ml or ● 2 x 5 ml to inoculate ▲ 2 x 100 ml or ● 2 x 500 ml LB+Cm25+Ara0.1% in ▲ 500 ml or ● 2500 ml flasks. Culture overnight at 37°C. Shake cultures vigorously – minimum 250 rpm.
Harvest the bacterial cells by centrifugation at 6000 x g for 15 min at 4°C.
Resuspend the bacterial pellet from both flasks combined in ▲ 8 ml or ● 50 ml of Buffer P1.
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.
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.
Centrifuge at ≥20,000 x g for 30 min at 4°C. Remove supernatant containing plasmid DNA promptly.
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.
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.
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.
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.
Apply the DNA solution from step 10 to the QIAGEN-tip and allow it to enter the resin by gravity flow.
Wash the QIAGEN-tip with ▲ 2 x 10 ml or ● 2 x 30 ml Buffer QC.
Elute DNA with ▲ 5 ml or ● 15 ml Buffer QF.
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.
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.
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.
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.
You should obtain in total ▲ 100 μg or ● 500 μg of pure injection-quality fosmid DNA.
This protocol has been posted on Protocol Exchange, an open repository of community-contributed protocols sponsored by Nature Portfolio. These protocols are posted directly on the Protocol Exchange by authors and are made freely available to the scientific community for use and comment.
posted
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