VIDEO I

 Verification of recombinant yeast strains

ii. isolaton of genomic DNA

Verifikasi strain ragi rekombinan

ii. isolasi DNA genomik

 buffers :1M Saccharose, 1M Tris HCL (pH 8,0)

- SE Buffer :0,2 % SDS, 50mM EDTA (pH 8,0)
- 3M Kac (PH 5,4)
- Ethanol 70%
- Isopropanol
- Lyticase
- dH2o

-penyangga: Saccharose 1M, 1M Tris HCL (pH 8,0)

- SE Buffer: 0,2% SDS, 50mM EDTA (pH 8,0)

- 3M Kac (PH 5,4)

- Etanol 70%

- Isopropanol

- Lyticase

- dH2o

 Transfer 1,5 ml from an overnight

Culture of yeast cells to a microfuge tubes.

- Transfer 1,5 ml dari semalam

Budaya sel ragi ke tabung microfuge.

 Pellet the cells by centrifunging at 4000 rpm

At room temperature in a microfuge.
- Pelet sel dengan sentrifugasi pada 4000 rpm
Pada suhu kamar di microfuge.
Tunggu 2 menit
 Remove the culture medium
- Hapus media kultur
 Rinse the pellet With 500 micro liters of ST buffer
- Bilas pelet Dengan 500 mikro liter ST buffer
 Vortex 3-5 times
Centrifugation at 4000 rpm 2 minute
Remove the supernatant Add 200 micro liters ST buff
- Vortex 3-5 kali

Sentrifugasi pada 4000 rpm 2 menit

Hapus supernatan Tambahkan 200 micro liter ST buffer

Vortex 3-5 kali

 Add 2 macro liters Lyticase(forvthe destruction of the cell wall)

Incubate at 28 celcius 1 hours Centrifugation at 4000 rpm. 2minute.

Remove the supernatant

- Tambahkan 2 liter makro Lyticase (untuk penghancuran dinding sel)

Inkubasi pada 28 celcius 1 jam Sentrifugasi pada 4000 rpm. 2 menit.
Hapus supernatan
 Resuspend the pellet in 500 micro lyters SE buffer (for the destruction of the nucleus)
Vortex 3-5 times Place in a bath at 70 celcius place in a water bath at 70 celcius 15 minute
Cool to room temperature
Add 300 micro lyters cold 3M KAC (ph 5,4)
(for precipitstion of proteins)
Cool at 20 celcius 15 minute.
- Resuspensi pelet di 500 micro lyters SE buffer (untuk penghancuran nukleus)
Vortex 3-5 kali Tempatkan dalam bak mandi di 70 celcius di air mandi dengan suhu 70 celcius 15 menit
Dingin hingga suhu kamar
Tambahkan 300 mikro lyters dingin 3M KAC (ph 5,4)
(untuk presipitstion protein)
Dinginkan pada 20 celcius 15 menit.
 Centrifigation at 4000 rpm. 2minute.
- Sentrifugasi pada 4000 rpm. 2 menit.
VIDEO II
 Centrifugation at 4000 rpm, 2 minute.

Transfer supernatant to new micro tubes

Add 600 micro lyters isopropanol (for DNA binding)

Centrifugation at 13 000 rpm, 10 minute

- Sentrifugasi pada 4000 rpm, 2 menit.

Transfer supernatan ke tabung mikro baru

Tambahkan 600 mikro lyters isopropanol (untuk DNA yang mengikat)

Sentrifugasi pada 13.000 rpm, 10 menit

 Remove the supernatant

Rinse the pellet With 1 ml of Ethanol 70 %
Centrifugation at 13000 rpm, 10 minute
Remove the supernatant
- Hapus supernatan

Bilas pelet Dengan 1 ml Etanol 70%

Sentrifugasi pada 13000 rpm, 10 menit

Hapus supernatan

 Spin down
Discarded remaining of supernatant
Dry at 37 celcius 15 minute
Dissolve the DNA in dh2O
- Putar ke bawah
Buang sisa supernatan
Keringkan pada 37 celcius 15 menit
Larutkan DNA dalam dh2O