18.104.22.168. Preparation of genomic DNA agarose plugs
- Culture an isolate of P. larvae on an MYPGP agar plate for 48 h at 37°C.
- Suspend a loopful of bacteria (2-3 colonies from an isolated area on the plate) into 500 µl MYPGP broth and centrifuge for 1 min at RT.
- Remove the broth, suspend the pellet with 1 ml washing buffer (see recipe below), and centrifuge for 1 min at RT.
- Remove the washing buffer.
- Completely suspend the bacterial cell pellet in 0.30 ml washing buffer + 0.05 ml Proteinase K (0.5 mg/ml, see recipe below).
- Warm the suspension to 50°C.
- Mix the suspension with an equal volume of melted 2% SeaKem Gold agarose (prepared in washing buffer; Cambrex Bio Science Rockland, Inc., ME) at 50°C.
- Quickly pipette into two wells of a plug mold (Bio-Rad Laboratories, Inc.) warmed to 37°C.
- Solidify the plugs at 4°C for 30 min.
- Remove the two plugs from the mold.
- Add plugs to 5 ml preheated Proteinase K Solution in a 50 ml plastic culture tube.
- Incubate overnight at 50°C with gentle agitation (shaker water bath).
- Preheat 10 ml H2O and 40 ml TE80 buffer (see recipe below) to 50°C.
- Wash plugs (use a sterile BioRad green screened caps; part #170-3711) with 10 ml 50°C H2O (add, swirl, and drain).
- Wash plugs with 10 ml 50°C TE80 buffer 4X (15 min for each wash with gentle shaking).
- Add 5 ml RT TE80 buffer to the plugs in the 50 ml culture tube and store 4°C. Plugs are good for approximately 2 months in TE80 buffer at 4oC.
Wash Buffer (100 ml):
- 200 mM NaCl (4.0 ml of 5 M stock)
- 10 mM Tris-HCl (pH 7.5) (1.0 ml of 1 M stock)
- 100 mM EDTA (20.0 ml of 0.5 M stock)
Proteinase K Solution (100 ml):
- 50 mM EDTA (10.0 ml of 0.5 M stock)
- g N-lauroylsarcosine
- 50 mg Proteinase K (final 0.5 mg/ml)
- 50 mg Lysozyme (final 0.5 mg/ml)
TE80 Buffer (100ml):
- 10 mM Tris-HCl (pH 8.0) (1.0 ml of 1 M stock)
- mM EDTA (0.2 ml of 500 mM stock)