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home lab www.mlang.org	Preparation of Nucleic Acids Genomic DNA extraction from 5-10 flies The protocol is suitable for DNA extraction from adults but also for DNA extraction from other developmental stages of Drosophila. It is designed for DNA extraction from a single individual, but it also can easily be adapted to other quantities (i.e. 1-5 Flies: 100µl Solution A; for 6-10 Flies: 200µl Solution A; up to 50 Flies: 500µl Solution A). *Solutions* KOAc [8M], filter sterilized (2ml aliquots, stored at-20°C, have to be defrosted 20min before use) Solution A (100mM Tris pH: 9.0, 1% SDS, 100mM EDTA) TE Buffer (Tris-HCl [10mM], EDTA [1mM], pH: 8,0). Phenol (equilibrated to pH8,0) Phenol: Choroform (1 volume phenol : 1volume Chloroform: Isoamylic Alcohol [24:1]) Chloroform (Chloroform: Isoamylic Alcohol [24:1]) NH4OAc [7,5M], filter sterilized Ethanol [100%] Ethanol [70%] Protocol 1. Flies are homogenized with tube homogenizer sticks in a 1,5ml reaction tube with solution A (100µl for 1-5 flies, 200µl for 6-10). 2. Samples are set to incubate for 20-30min at 65°C and then left to equilibrate to room temperature. 3. A total of 14µl KOAc [8 M] is added per 100µl of the sample volume. The solutions are mixed by tipping slightly against the tube and are then placed on ice for 30min. 4. Samples are centrifuged for 10min at °C at 14000rpm in a table top centrifuge. The supernatants are transferred to a fresh tubes. This step is repeated once. 5. To each sample 2µl RNase A [10 mg/ml] is added and samples are put to incubate for 15min at 37C. 6. Sample volumes are adjusted to 300µl with TE Buffer and 300µl (1 vol.) phenol is added. The phases are mixed, samples are centrifuged samples for 5min at RT, 14.000rpm and the aqueous phase is transferred to a fresh 1,5ml tube. This extracton step is repeated with 1 volume phenol:chloroform and once with 1 volume chloroform. 7. Finally, the aqueous phase is recovered into fresh 1,5ml tubes and DNA precipitated from each sample by addition of 0,5 volumes NH4OAc [7,5M] and 2.5 volumes of cold ethanol [100%] were added. Samples are stored overnight at -20°C. 8. DNA from each sample is pelleted by centrifugation for 15min at 14000rpm at 4°C. Supernatant are discarded and DNA pellets are washed with 200µl ethanol [70%]. Samples are centrifuged once more for 5min at 14000rpm at 4°C, supernatant are removed and pellets are left to dry. Pellets are resuspended in 20µl TE Buffer and 1µl of resuspended DNA solution is examined on an 0,8% agarose gel.

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Preparation of Nucleic Acids

Genomic DNA extraction from 5-10 flies

The protocol is suitable for DNA extraction from adults but also for DNA extraction from other developmental stages of Drosophila. It is designed for DNA extraction from a single individual, but it also can easily be adapted to other quantities (i.e. 1-5 Flies: 100µl Solution A; for 6-10 Flies: 200µl Solution A; up to 50 Flies: 500µl Solution A).

Solutions

KOAc [8M], filter sterilized (2ml aliquots, stored at-20°C, have to be defrosted 20min before use)
Solution A (100mM Tris pH: 9.0, 1% SDS, 100mM EDTA)
TE Buffer (Tris-HCl [10mM], EDTA [1mM], pH: 8,0). Phenol (equilibrated to pH8,0)
Phenol: Choroform (1 volume phenol : 1volume Chloroform: Isoamylic Alcohol [24:1])
Chloroform (Chloroform: Isoamylic Alcohol [24:1])
NH4OAc [7,5M], filter sterilized
Ethanol [100%]
Ethanol [70%]

Protocol

1. Flies are homogenized with tube homogenizer sticks in a 1,5ml reaction tube with solution A (100µl for 1-5 flies, 200µl for 6-10).

2. Samples are set to incubate for 20-30min at 65°C and then left to equilibrate to room temperature.

3. A total of 14µl KOAc [8 M] is added per 100µl of the sample volume. The solutions are mixed by tipping slightly against the tube and are then placed on ice for 30min.

4. Samples are centrifuged for 10min at °C at 14000rpm in a table top centrifuge. The supernatants are transferred to a fresh tubes. This step is repeated once.

5. To each sample 2µl RNase A [10 mg/ml] is added and samples are put to incubate for 15min at 37C.

6. Sample volumes are adjusted to 300µl with TE Buffer and 300µl (1 vol.) phenol is added. The phases are mixed, samples are centrifuged samples for 5min at RT, 14.000rpm and the aqueous phase is transferred to a fresh 1,5ml tube. This extracton step is repeated with 1 volume phenol:chloroform and once with 1 volume chloroform.

7. Finally, the aqueous phase is recovered into fresh 1,5ml tubes and DNA precipitated from each sample by addition of 0,5 volumes NH4OAc [7,5M] and 2.5 volumes of cold ethanol [100%] were added. Samples are stored overnight at -20°C.

8. DNA from each sample is pelleted by centrifugation for 15min at 14000rpm at 4°C. Supernatant are discarded and DNA pellets are washed with 200µl ethanol [70%]. Samples are centrifuged once more for 5min at 14000rpm at 4°C, supernatant are removed and pellets are left to dry. Pellets are resuspended in 20µl TE Buffer and 1µl of resuspended DNA solution is examined on an 0,8% agarose gel.