Miniplasmid Prep for ABI Sequencing or Restriction Analysis
1) Pick colonies from lZAPII excision or transformations (ie. after preparation of nested deletions) onto a grid plate. For this purpose, place an LB amp plate onto a 50 subdivision grid (held by pins; mark one spot on perimeter of plate bottom to correspond to mark on grid for orientation). Using sterile toothpicks, pick individual colonies and touch toothpick first to grid plate and then immerse in 2 ml of LB broth or Terrific broth containing 2 l of stock 50 mg/ml ampicillin (use 2059 tube). Pick 10 colonies per excision or deletion time point. Do a LB or Terrific broth inoculation for only 1-3 colonies per same sample. Incubate overnight at 37°C.
2) Pellet 1.5 ml aliquots for 1 min in Eppendorf microcentrifuge. Discard media and resuspend pellet in 200 l of GTE (to 1086 l of ddH2O add 60 l of 1 M glucose, 30 l of 1 M Tris, pH 8, and 24 l of 0.5 M EDTA) by pipetting up and down. Add 300 l of freshly prepared 0.2 N NaOH/1% SDS (to 1209 l of ddH2O add 26 l of 10 N NaOH and 65 l of 20% SDS) and mix by inversion. Incubate on ice for 5 min.
3) Neutralize by adding 300 l of 3 M (or 180 l of 5 M) potassium acetate, pH 4.8, mix by inversion and incubate on ice for 5 min. (For 3 M potassium acetate, make 29.4 gm of potassium acetate up to a final volume of 60 ml in ddH2O. Add 11.5 ml of glacial acetic acid and 28.5 ml of ddH2O; autoclave).
4) Spin for 10 min at RT. Transfer supernatant to a fresh tube and add 27.2 l of 0.5 mg/ml RNase (DNase-free; Boehringer Mannheim #1119919). Incubate for 20 min at 37°C.
5) Extract supernatant with 400 l each of chloroform. Mix by hand for 30 sec, spin for 1 min. Carefully remove aqueous phase. Reextract with 400 l of chloroform. Extract a third time if debris is visible after second extraction.
6) Precipitate DNA by adding an equal volume of 100% isopropanol. Spin immediately for 10 min at RT. Wash pellet with 500 l of 70% ethanol. Air dry and dissolve pellet in 62.7 l of ddH2O. Precipitate plasmid DNA by adding 17.3 l of 30% PEG/2.5 M NaCl, mix by inversion. Incubate on ice for 20 min and pellet by centrifugation for 15 min at 4°C.
7) Remove supernatant and rinse pellet with 500 l of 70% ethanol. Air dry and resuspend in 20 l of ddH2O. Determine OD260 by diluting 5 l up to 500 l in ddH2O. OD260=1.0 is 50 g/ml. OD260/OD280=2.0 for DNA. For restriction analysis, linearize by digesting 1 l of plasmid in a 20 l reaction volume for 1 hr at 37°C. Add 4 l of gel sample buffer and load. Examine in a midsize 0.9% agarose gel (100 ml containing 0.9 gm agarose; 5 l of ethidium bromide) ran for 1 hr at 150 V. Prior to sequencing, do Southern analysis (run EcoRI [or suitable enzyme that will remove insert] digest on gel, transfer and hybridize with random primed 32P-gliadin cDNA [or with labeled oligo]) and Northern analysis (run poly A+ RNA from different tissues including lacrimal acinar cell on gel, transfer and hybridize as above) to select clones. For ABI sequencing, take 0.5 g up to 8 l in ddH2O.