Manual protocol

Safety procedure

Use for the protocol pipet tips with filters

Materials

• 0.35 M sorbitol
• 0.1 M Tris-HCL, pH9
• 5 mM EDTA, pH8
• 2 M NaCl
• 2% CTAB (Cetrimonium bromide)
• Sarkosyl (N-lauroylsarcosine sodium salt)
• Polyvinylpyrrolidone (PVP) (40000 MW) 1 % [w/v]
• RNAse T1
• Potassium Acetate 5M (KAc precipitate polysaccharides) pH 7.5
• Buffered Phenol:Chloroforme:Isoamylalcool P:C:I (25:24:1)

Equipment

• 50 ml vails

Buffer A

• 0.35 M sorbitol
• 0.1 M Tris-HCL, pH9
• 5 mM EDTA, pH8

Autoclave to sterilize

Buffer B

• 0.2 M Tris-HCL, pH9
• 50 mM EDTA, pH8
• 2 M NaCl
• 2% CTAB

Autoclave to sterilize

Buffer C

• 5% Sarkosyl

Mix in 65 C water bath Transfer trough 0.22 u filters

Protocol

1. Mix buffers A+B+C(2.5:2.5:1 + 0.1%PVP final)- lysis buffer

• Close the air-condition when working with PVP
• Mix in 65 C water bath
• Cool in room temperature
• Use 50 ml vails
• Use 17.5 ml for each reaction

1. Add to the lysis buffer (17.5 ml) 10 ul (10kU) of RNAse T1
2. Crush the samples in sand (with mortar and pestle) - 1 gr of sand for 100 mg of sample

• Crush for 2 min and add 15 ml liquide nitrogen every 15 sec

1. Transfer the powder the 50 ml vail with 17.5 ml lysis buffer and vortex
2. Keep in room temperature for 30 min (invert sides every 5 min)
3. Add 200 ul Proteinase K (according to 800U/ml), Kepp in room temperature for 30 min and invert sides every 5 min
4. Cool in ice for 5 min
5. Add 3.5 ml KAc, shake gently and put in ice for 5 min

• Don't leave over ice for too long

1. Spin in 4c at 5000g for 12 min
2. Transfer the supernatant to clean plastic tune with 17.5 ml P/C/I