1. Spin down about 8x107 live cells (this is about 11.5 ml of an OD660=0.5UA/ml culture and enough to make just 1 plug). 2500g x 1 min.
2. Resuspend in 1 ml 50 mM EDTA and transfer to microfuge tube.
3. Spin down cells by centrifuging for 10 seconds at top speed.
4. Wash once more time with 1 ml 50 mM EDTA.
5. Add 10 μl 50 mM EDTA and keep on ice.
6. Melt 1% Low Melting Point (LMP) agarose solution and store at 50ºC.
7. Tape up the bottom of clean, dry plug formers. Place on ice.
8. Add 20 μl of Solution 1. Keep on ice.
9. Mix 60 μl of molten LMP agarose solution, mix by pipetting up and downand dispense into plugs formers on ice.
10. Push out plugs into a fresh labelled 2 ml microfuge tube. Add solution 2to fill up the tube.
11. Gently invert a few times to mix.
12. Incubate overnight at 37ºC.
13. Chill the tubes on ice for 10 min.
14. Remove solution 2.
15. Add solution 3 and incubate overnight at 37ºC.
16. Remove solution 3.
17. Add storage buffer and store -20ºC.
18. Before storing or loading the PFGE, treat plugs with Pefabloc 1 mg/ml(diluted in water) for 1 h and wash with TE if you are going to treat the plugs with restriction enzymes or similar.
Solutions:
• SCE: 1 M sorbitol, 0.1 M sodium citrate, 0.06 M EDTA.
• 1% LMP agarose solution: 1% LMP agarose, 0.125 M EDTA.
• Solution 1: 5 μl/ml β-mercaptoethanol + 1 mg/ml zymolyase 100T (100000 units/g; stock: 4 U/μl) in SCE
• Solution 2: 0.45 M EDTA, 0.01 M Tris-HCl pH 7.5, 7.5% β-mercaptoethanol, 10 μg/ml RNaseA.
• Solution 3: 0.25 M EDTA, 0.01 M Tris-HCl pH 7.5, 1% sarkosyl, 1 mg/ml proteinase K.
• Storage solution: 0.05 M EDTA, 50% glycerol.