Preparasi DNA fag

Tutorial bioteknologi
 Bacteriophages, or phages as they are commonly
known, are viruses that specifically infect bacteria.

 Like all viruses, phages are very simple in structure,

consisting merely of a DNA (or occasionally
ribonucleic acid (RNA)) molecule carrying a number
of genes, including several for replication of the
phage, surrounded by a protective coat or capsid
made up of protein molecules
 The general pattern of
infection of a bacterial
cell by a bacteriophage
 Phage DNA infection
M13 phage DNA infection
 DNA fag dapat keluar dari sel dengan
sendirinya. Tidak perlu dilakukan
prosedur ekstraksi sel
 Selanjutnya dilakukan sentrifugasi.
DNA fag tersuspensi
 Kesulitannya terletak pada bagaimana
mendapatkan DNA fag dengan jumlah
tinggi dalam tiap mL kultur
Meningkatkan
jumlah fag
 Pastikan fag dalam
fasa lisis
 cI menjaga fag tetap
dalam fasa lisogenik
 Mutan cIts
(temperature sensitive)
akan masuk dalam
fasa lisis pada 42oC
 Fag harus menginfeksi
bakteri tepat pada saat
siklus pertumbuhan

Induction of a l cIts lysogen by

transferring from 30°C to 42°C.
Achieving the right balance between culture age and inoculum
size when preparing a sample of a non-lysogenic phage.
 Fag dapat diendapkan dengan
polyethylene glycol (PEG) + NaCl
 Disentrifugasi. Supernatan dikeluarkan
dan pelet (residu) dilarutkan kembali.
 Pemurnian dilakukan dengan Gradien
densitas CsCl
Collection of phage particles by polyethylene glycol (PEG) precipitation.
Purification of l phage particles by CsCl density gradient centrifugation.
Preparation of single-stranded M13 DNA from an infected culture of bacteria.