The plug was treated with TNE buffer (10 mM Tris HCl, 50 mM EDTA, 1 M NaCl, pH 8.0) containing 0.2% sodium lauroylsarcosinate (Nacalai Tesque), 0.2% sodium deoxycholate (Nacalai Tesque), 2 mg/ml lysozyme (Nacalai Tesque), and 2.5 µg/ml ribonuclease A at 37 °C overnight, followed by treatment with 1% sodium dodecyl sulfate and 100 µg/ml proteinase K overnight.
The rest of the plug was returned to long term storage in TE buffer.
Pulling the plug is unwise.
After being washed 3 times with TE (Tris 0.5M EDTA) buffer at 37°C, the plugs were restricted with 20 U of NheI (New England Biolabs, Beverly, MA), 330 μg/mL of bovine serum albumin, and 200 μL of New England buffer #2 at 37°C overnight.
The plugs were incubated in lysis buffer (TNE buffer, 1% SDS, 1 mg/ml proteinase K, 0.25% TritonX-100) for 24 h twice at 50°C, washed with TNE buffer, and inactivated with TNE buffer supplemented with phenylmethylsulfonyl fluoride (0.04 mg/ml) at 50°C for 1 h.
The plugs were stored in TE buffer at 4°C and used within two months.
Then, the plugs were stored in TE buffer (pH 8.0) at 4°C for future use.
Once solidified the plugs were placed in a buffer containing 5 ml lysis buffer and 200 μl lysozyme (from a stock solution of 20 mg/ml; Roche Diagnostics Scandinavia AB, Bromma, Sweden) and gently shaken overnight at 37°C.
The plugs were rinsed twice in TE buffer for 30 min and stored in TE buffer at 4°C.
The plugs were lysed in lysis buffer supplemented with RNase (5 mg/mL) and lysozyme (1 mg/mL) which was incubated overnight, followed by fresh lysis buffer with proteinase K (0.5 mg/mL) at 50°C for 48 hours.
