Manual protocol

Safety procedure

• Use for the protocol pipet tips with filters
• After adding the P/C/I work in the hood

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)
• RNAse T1 (1000 U/ml)
• Sodium Acetate (NaAc) 3M pH 5.2
• Isopropanol

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 liquid 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), Keep 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 (the white strings are the DNA)

• Don't leave over ice for too long

1. Spin in 4c at 5000g for 12 min
2. Transfer the supernatant to clean plastic tube with 17.5 ml P/C/I (from this stage to work in the hood and dont do vortex)
3. Mix gently by turning upside-down the tube. with Three fazes will be created - the upper faze is what we want - it is better to take less than to take other fazes
4. Spin of 4 degrees with 4000g for 10 min
5. Transfer the supernatant to clean plastic tube with 5 ul RNAse T1 (to dissolve RNA work with filtered tips)
6. Incubate for 20-30 min in room temperature
7. Add NaAc (0.1 of total volume ~1.8 ml), turn upside-down - make sure the added volume is 0.1)
8. Add Isopropanol (1 of total volume ~18 ml), turn upside-down - make sure the added volume is 1)
9. Incubate in room temperature for 10 min
10. Spin of 4 degrees with 10,000g for 30 min (to submerge the DNA)
11. Carefully remove the supernatant for 1-2 ml will remain (contain the DNA) - use cut tip, so the DNA will not be harmed
12. transfer the pellet to clean ephendorf tube (if the DNA disappear, add 1.5 ml of EtOH 70%, spin down in 4000g for 5 min, remove 1 ml from the supernatant and collect the DNA to clean tube)
13. Spin in 4 degrees with 13,000g for 5 min (to submerge the DNA)
14. carefully remove the supernatant and wash with 1.5 ml of EtOH 70%, shake upside-down slowly
15. Spin in 4 degrees with 13,000g for 5 min and remove supernatant (reapet the washing two times)