Prosedur Isolasi Dna_biotek_2016_d.pdf

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Siapkan: 1. Lysozyme 20 mg/ml 2. Dapar TE: 10 mM Tris HCl + 1 mM EDTA

Sentrifugasi suspensi bakteri 6000 rpm 5'

Buang supernatan sekering mungkin

Resuspensi dengan 100 µL dapar TE+2 µL lysozyme

Campur dengan pipetting

Tambah 450µL lysis solution RL

Campur dengan pipetting

Inkubasi 3' suhu ruang

sentrif kecepatan maks 1'

pindahkan supernatan ke spin filter D

Masukkan ke collection tube

sentrif 12000 rpm 2'

ganti collection tube

tambahkan 500 µL washing solution HS

sentrif 12000 rpm 1'

Buang filtrat

tambahkan 700 µL washing solution LS

sentrif 12000 rpm 1'

Buang filtrat

sentrif 12000 rpm 2'

Pindahkan spin filter ke elution tube

Tambahkan 100µL elution buffer

Inkubasi suhu ruang 1'

sentrif 8000 rpm 1'

simpan di 20°C

Persiapan suspensi bakteri (aslab)

Koloni E coli dari cawan 18 jam

Untuk 1 kelompok

inokulasi ke 100 ml LB cair

Inkubasi 18 jam 37°C shaker 200-250 rpm

Pindahkan 1.5 ml biakan ke tabung 1.5 ml

sentrif 6000 rpm 5'

buang supernatan

tambah 1 ml suspensi

buang supernatan

cuci dengan 1 ml aquabidest

sentrif 6000 rpm 5'

buang supernatan

Ulangi 2 kali

sentrif 6000 rpm 5'

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