Authors: Radoslaw Kamil Ejsmont
Introduction
This is a quick and robust protocol for isolation of High Molecular Weight genomic DNA from Drosophila embryos. We have used DNA isolated using this protocol for fosmid genomic library production.
Procedure
- Collect embryos for 24h. Optional: Let them age for up to 12h at room
temperature.
- Decorionate embryos for 2 minutes in 100% bleach fluid (2% sodium
perchloride).
- Wash embryos with 1 x PBS.
- Wash embryos with 1 x PBT.
- Transfer embryos into a bottle containing 1 volume of PBS and 1 volume of n-
Heptane. Use 20 ml of PBS per 1 ml of embryos. Mix by briefly shaking the
bottle.
- Remove PBS (lower phase). Leave the interphase intact.
- Add 1 volume of methanol and shake vigorously by hand for 1 minute.
- Remove n-heptane and interphase.
- Transfer embryos into the Falcon tube (15ml or 50ml depending on the
sample volume) and wash twice with 1 volume of methanol.
- Remove methanol.
- Wash embryos with 1 volume of PBS.
- Add 1 volume of lysis buffer (50 mM Tris-HCl pH 8.0, 100 mM EDTA, 100 mM
NaCl, 0.5% SDS, 5 mg/ml Proteinase K, 100 mg/ml RNAse A).
Lyse for 2-3 hours at 55°C. Gently mix by inverting the tube every 15 minutes.
- Centrifuge at 4000g for 15 minutes. Transfer supernatant to a new Falcon
tube. Optional: Remove 200 μl for quality analysis.
- Add 1 volume of Phenol:Chloroform:Isoamyl alcohol. Incubate on
a rotating wheel for 1 hour at 4°C.
- Centrifuge at 4000g for 10 minutes. Transfer aqueous (upper) phase to
a new Falcon tube.
- Repeat steps 14–15.
- Add 1 volume of Chloroform:Isoamyl alcohol. Incubate on a rotating wheel for
1 hour at 4°C.
- Centrifuge at 4000g for 10 minutes. Transfer aqueous (upper) phase to
a new Falcon tube.
- Add 0.05 volume of 3.0 M KAc. Mix by gently inverting the tube.
- Add 0.7 volume of isopropanol. Incubate on a rotating wheel for 30 minutes at
4°C.
- Centrifuge at 6000g for 15 minutes. Remove supernatant.
- Wash the pellet twice with 1 volume of 70% ethanol.
- Air-dry the pellet for 10 minutes at room temperature.
- Dissolve the pellet in 1 x TE prewarmed to 55°C. Store DNA at 4°C.
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.2009.107. Originally published online 27 May 2009.