Drosophila sequencing method for 96-well plates
1. Collect ~15 flies into each well of a 1.2ml mircotitre plate (ABgene). Keep the plate on ice while transferring the flies to keep them asleep / lethargic.
1. Switch on 65°C water bath.
Restriction of genomic DNA
1. Set up the following reaction in a 0.2ml PCR plate (ABgene). Use Roche Msp1.
2. Seal plate with transparent adhesive seal.
3. Incubate 3hr at 37°C, and then 40min at 65°C in the PCR machine.
4. Run 5ul of reaction on a 1% agarose gel to check that the reaction has worked.
Ligation of restricted DNA
1. Set up the following reaction in a 1.2ml microtitre plate:
2. Incubate plate at RT for 2hr.
3. Add 20ul 3M NaAc and 500ul 96% EtOH to each well and mix.
4. Incubate -20°C for 20min.
5. Spin plate 4000rpm 30min.
6. Decant supernatant and wash pellet in 500ul 70% EtOH.
7. Spin plate 4000rpm 15min.
8. Decant supernatant and dry pellet.
9. Re-suspend pellet in 50ul MQ (Sigma).
1. Set up the following reaction in a 0.2ml PCR plate (ABgene):
2. Run PCR program BDGP-P (PRY, RS3 55°C anneal ) or BDGP_2 (PLAC 60°C anneal):
95°C 15min 95°C 30sec } Anneal °C 1min } x35 cycles 72°C 2min } 72°C 10min3. Run 5ul of product on a 2% agarose gel to check reaction has worked.
Purification of PCR products
1. Purify products using a Millipore Multiscreen PCR Purification plate.
Sequencing of purified PCR products
1. Set up the following reaction in a non-skirted 0.2ml PCR plate:
2. Perform sequencing reaction in PCR machine using program SEQ.CYC (standard cycling method from protocol).
3. Add 25ul ice cold 96% EtOH and 1ul NaAc (Applied Biosystems) to each sample and mix.
4. Precipitate at room temperature for 15min exactly.
5. Spin plates at 4000rpm 30min.
6. Decant supernatant and wash pellets in 125ul 70% EtOH.
7. Spin plates 4000rpm for 15min.
8. Decant supernatant and spin plates upside-down on a tissue at 250rpm for 1min.
9. Make sure that no liquid remains in the tubes, or unincorporated terminators will be carried over.
10. Dry pellets, cover plate and store at -20°C until needed, or add 10ul HiDi formamide loading buffer and run on the ABI3100 sequencer immedia tely.
RS3-1A TTATGAGTTAATTCAAACCCCAC RS3-2 TACGTACTCGCGATGAGCAC PRY1 CCTTAGCATGTCCGTGGGGTTTGAAT PRY4 CAATCATATCGCTGTCTCACTCAPrimers for amplifying up the 5' end of the RS elements:
RS5F CGTACTTTGGAGTACGAAATGC RS5R CGAATCATTAAAGTGGGTATCAC PLAC1 CACCCAAGGCTCTGCTCCCACAAT PLAC4 ACTGTGCGTTAGGTCCTGTTCATTGTT
PRY and PLAC primers are from the Berkeley Drosophila Genome Project at http://www.fruitfly.org/about/methods/inverse.pcr.html