DNA Extraction (guanidine isothiocyanate}
1. Prepare 2-3 ml EDTA-blood sample in 15 ml Falcon tube
2. Add eritrosit lysis buffer up to 5x dilution (8-12 ml) cat: tambah EL buffer ad 5 x misal 2 ml tambal 10 mL
3. Invert several times gently to mix the solution
4. put on ice for 20 minute
5. spin at 750 g for 10 minute
6. discard supernatan; clean up the tube wall by invert (dimiringkan) falcon tube in tissue paper
7. add 100 uL of SE buffer
8. replace solution into of SE buffer (campur dengan menyedot beberapa kali)
9. add 100 uL of guanidine isothyocyanate 4 Molar, mix well by pippeting
10. add 700 uL of chloroform
11. add 400 uL of NaCl 6 Molar
12. Shake strongly by hand (a critical step) 2-3 menit sampai timbul gumpalan yg tetap)
13. spin at 10000 rpm for 10 min
after sentrifugation, 3 layer appear in the solution ( transparent in upper (DNA0 and lower part , whereas milk like in the middle part/at wall of the tube (protein)
14. replace upper part of solution in a new1,5 mL tube. use pipet and note the volume of solution
15. add 1 volume isopropanolol/ethanol absolute solution ( 1:1= larutan: propanolol). shake gently by hand. DNA appears in the solution as a string or cloudy precipitation (this step is called isopropanolol/ethanol precipitation)
16. spin at 10.000 rpm for 2 min after centrifugation, DNA pellet is formed
17. discard supernatan
Tahap pencucian
18. add 500 ul of ethanol 70%; shake gently by hand
19. spin at 10,000 rpm for 5 minutes
20. discard supernatant
21. wipe the left liquid at the wall using tissue papper. don't touch DNA pellet
22. dry up at room temperature for 15 minutes (or at 55 C for 4-5 minutes)(suhu ruang dengan tutup terbuka)
23. add TE buffer 100-400 uL (depend on pellet volume)
24. store the sample at 4 derajat Celcius overnight and use in the PCR experiment (simpan disuhu -20 C sampai digunakan
Metode ekstraksi ada macam-macam:
1. phenol
2. Guanidine isotiocianat
3. salting out
4. silica gel
Prinsip isolasi
1. preparasi jaringan
2. lisis organel sel
3. denaturasi
4. presipitasi
5. washing
2. Add eritrosit lysis buffer up to 5x dilution (8-12 ml) cat: tambah EL buffer ad 5 x misal 2 ml tambal 10 mL
3. Invert several times gently to mix the solution
4. put on ice for 20 minute
5. spin at 750 g for 10 minute
6. discard supernatan; clean up the tube wall by invert (dimiringkan) falcon tube in tissue paper
7. add 100 uL of SE buffer
8. replace solution into of SE buffer (campur dengan menyedot beberapa kali)
9. add 100 uL of guanidine isothyocyanate 4 Molar, mix well by pippeting
10. add 700 uL of chloroform
11. add 400 uL of NaCl 6 Molar
12. Shake strongly by hand (a critical step) 2-3 menit sampai timbul gumpalan yg tetap)
13. spin at 10000 rpm for 10 min
after sentrifugation, 3 layer appear in the solution ( transparent in upper (DNA0 and lower part , whereas milk like in the middle part/at wall of the tube (protein)
14. replace upper part of solution in a new1,5 mL tube. use pipet and note the volume of solution
15. add 1 volume isopropanolol/ethanol absolute solution ( 1:1= larutan: propanolol). shake gently by hand. DNA appears in the solution as a string or cloudy precipitation (this step is called isopropanolol/ethanol precipitation)
16. spin at 10.000 rpm for 2 min after centrifugation, DNA pellet is formed
17. discard supernatan
Tahap pencucian
18. add 500 ul of ethanol 70%; shake gently by hand
19. spin at 10,000 rpm for 5 minutes
20. discard supernatant
21. wipe the left liquid at the wall using tissue papper. don't touch DNA pellet
22. dry up at room temperature for 15 minutes (or at 55 C for 4-5 minutes)(suhu ruang dengan tutup terbuka)
23. add TE buffer 100-400 uL (depend on pellet volume)
24. store the sample at 4 derajat Celcius overnight and use in the PCR experiment (simpan disuhu -20 C sampai digunakan
Metode ekstraksi ada macam-macam:
1. phenol
2. Guanidine isotiocianat
3. salting out
4. silica gel
Prinsip isolasi
1. preparasi jaringan
2. lisis organel sel
3. denaturasi
4. presipitasi
5. washing
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