Virginie Orgogozo
Quick Fly genomic DNA prep
(BDGP protocol)
Tris-HCl 1M pH=9.2 (150mL):
18.16g of Tris-Base in 80mL distilled water, mix, add about 10mL of concentrated HCl until pH 9.2. Adjust volume to 150mL.
buffer A (100mL):
10mL Tris-HCl 1M pH 9.5
3.7g EDTA dissodium
0.58g NaCl
0.5g SDS
in 70mL water. Adjust pH with NaOH 10N to pH 7.5.
Adjust volume to 100mL.
KAc 5M (100mL):
49.1g KAc in 80mL water, adjust volume to 100mL.
LiCl 6M (200mL):
72.49g LiCl in 150mL water, adjust volume to 200mL.
1. put the fly in an Eppendorf tube.
2. freeze at -80∞C (at least 10min).
3. grind the fly in 200uL buffer A+5uL RNAse (20mg/mL) with disposable tissue grinder.
4. add an additional 200uL buffer A and continue grinding until only cuticles remain.
5. incubate at 65∞C for 30min.
6. prepare 300uL KAc + 750uL LiCl. Add 800uL of the LiCl/KAc solution and incubate on ice for at least 10min.
7. spin for 15min at RT.
8. transfer 1mL of the supernatant into a new tube, avoiding floating crud. (If crud transfers, respin)
9. add 600uL isopropanol, mix, spin 15min at RT.
10. aspirate away supernatant, pulse, aspirate, wash with 70% ethanol, dry.
11. resuspend in 50uL TE.
12. store at -20∞C.