Plasmid rescue

Genomic DNA was purified from 25 flies using the QIAamp kit (QIAGEN) according to manufacturer protocol. The plasmid rescue was done as previously described (Hamilton and Zinn, 1994).

Each pGawB insertion is rescued with 2 to 5 different restriction enzymes (EcoRI, HindIII, NotI, SacI and SalI). At least 6 clones are analysed by restriction digest to confirm the presence of a single P element insertion. When all the clones have the same digestion pattern, 2 of them are sequenced with the DJ51 oligo (5'-GAGTTAATTCAAACCCCACGG-3'). The T7 oligo (5'-GTAATACGACTCACTATAGGGC-3') is also used for clones rescued by SalI and SacI. In some instance we found clones not strictly identical, however after sequencing they always corresponded to the same genomic localization (restriction polymorphism resulted from incomplete genomic digest or bi-molecular ligation events).

Detailed protocols

QIAamp genomic DNA preparation




Plasmid rescue

Restriction digest
- 80µl genomic DNA is digested overnight with 60 units restriction enzyme (Boehringer) in a 100µl reaction volume
- Restriction enzyme is inactivated by heating 10mn at 75C
Ligation
- 439µl H2O, 60µl Ligase buffer 10x and 1µl T4 Ligase (NEB, 400u/µl) is added and the ligation reaction performed overnight at 16-18C
- DNA precipitation is done by adding 24µl LiCl 4M then 600µl isopropanol
- let stand on ice 30min
- centrifuge 10min 13000rpm
- pellet is washed with EtOH 70% and resuspended in 10µl H2O
Bacterial transformation
All steps before electroporation carried on in ice in a cold room
- 5µl Ligation reaction is mixed with 40µl Electrocompetent E.coli XL1Blue (10e9 transformant/µg control plasmid DNA, protocol to prepare electrocompetent cells)
- Bacteria and DNA solution is transfered into a 1mm Electroporation cuvette
- Electroporation 1.5V with GenePulser (Biorad) and immediate addition of 0.5ml Terrific Broth media
- Transfer to culture tube (Falcon 14ml) and incubate 30-60min 37C with agitation
- Plate the whole reaction on ampicillin bacterial plate
- Incubate overnight 37C
We obtain between 30 and 500 colonies, 6-12 colonies are analysed with 2 restriction enzymes (the one used for genomic DNA digestion and one to compare pattern between clones), at least 2 clones are sequenced.