6DETERGEN Melarutkan lipid pada membran sel CTAB (hexadecyltrimethylammonium bromide) – sel tumbuhanLaurylsarcosine—bakteri gram negative
7DETERGEN Soap molecules and grease molecules are made of two parts: Heads, which like waterTails, which hate water.
8DETERGENSel mempunyai membran lipid double layer dan protein.
9DETERGENWhen detergent comes close to the cell, it captures the lipids and proteins.
10Enzim protease—Proteinase K Menghilangkan nuclear protein, enzimMemecah ikatan peptidaBisa ditambahkan atau tidak (optional)PanasPaling sering 40-60ºCBufferTris HCl pH 8 untuk menjaga stabilitas DNA
112. Penghilangan protein & RNA a. Ekstrak sel dicampur dengan fenol, perlahan!b. Tambahkan Rnase pada lapisan aqueous
123. Pemekatan konsentrasi DNA Sentrifugasi70% final conc.“spooling”Ethanol precipitation
13ANALISIS KUANTITATIF DNA Absorbansi/Optical density (OD): c x b x (extinction coefficient, E).Asam nukleat mengabsorbsi sinar UV pada 260 nm 1 A260 O.D. unit for dsDNA = 50 µg/ml 1 A260 O.D. unit for ssDNA = 33 or 50 µg/ml 1 A260 O.D. unit for RNA = 40 µg/ml
15Double-stranded and single-stranded DNA differ in their optical absorption at 260 nm dCdGdUdsDNAssDNAnucleotidesThe conjugated p-electron systems of the purine & pyrimidine bases absorb strongly in the UV. (That’s why UV light is mutagenic and carcinogenic.)The absorbance of double-stranded DNA (dsDNA) at 260 nm is less than that of either single-stranded DNA (ssDNA) or the free bases. This is called “hypochromism.”
16Kemurnian DNAThe purity of the DNA is reflected in the OD260:OD 280 ratio and must be between 1.6 and 2.00.Decreased 260:280 ratio means that contaminating protein is still present.
17ANALISIS KUALTITAIF Metode: Elektroforesis Gel Agarose buffer Add enough electrophoresis buffer to cover the gel to a depth of at least 1 mm. Make sure each well is filled with buffer.Cathode(negative)Anode(positive)wells
19Agarose is a linear polymer extracted from seaweed. D-galactose3,6-anhydroL-galactoseSweetened agarose gels have been eaten in the Far East since the 17th century.Agarose was first used in biology when Robert Koch* used it as a culture medium for Tuberculosis bacteria in 1882*Lina Hesse, technician and illustrator for a colleague of Koch was the first to suggest agar for use in culturing bacteriaAgarose is a linear polymer extracted from seaweed.PRAKTIKUM BIOLOGI MOLEKULER, 2007
20+ - DNA H O2 Power • DNA is negatively charged. • When placed in an electrical field, DNA will migrate toward the positivepole (anode).HO2• An agarose gel is used to slow the movement of DNA and separate by size.+-PowerScanning Electron Micrograph of Agarose Gel (1×1 µm)• Polymerized agarose is porous,allowing for the movement of DNA
21+ - How fast will the DNA migrate? DNA small large Power strength of the electrical field, buffer, density of agarose gel…Size of the DNA!*Small DNA move faster than large DNA…gel electrophoresis separates DNA according to sizeDNA+-PowersmalllargeWithin an agarose gel, linear DNA migrate inversely proportional to the log10 of their molecular weight.
25Teknologi dna rekombinan Agustina Setiawati, M.Sc., Apt
26KloningDNA Cloning – the act of making many identical copies of a particular piece of DNA (often a gene)As you know, the first stop often involves joining a piece of DNA of interest to a cloning vector using DNA ligase
27Prinsip DNA rekombinan Penyisipan DNA target ke dalam vektorMemasukkan DNA rekombinan dalam bakteri
28TAHAP KLONING Pemilihan vektor Pemotongan vektor dan DNA target dengan enzim retriksiPenyambungan DNA pada vektorTransformasiSeleksi hasil transformasi
48ElectroporationPrinsip: pembukaan membran pembentukan pori sel tanaman dengan muatan listrikDNA in the surrounding solution can enter the cell through these pores and become incorporated into the cell’s nuclear genome through illegitimate recombination
49Biolistic transformation – “Gene gun” DNA is precipitated on the surface of heavy metal (gold; tungsten) particlesLoaded particles are driven into plant cells by high velocity gas propulsion (originally gunpowder; now helium)Distance between discharge nozzle and tissue can be optimized, as can particle velocityTarget tissue must be regenerable
54Seleksi pUC18/19Sel yang membawa plasmid (non rekombinan dan rekombinan) resisten terhadap ampisilin.Sel yang punya plasmid non rekombinan menghasilkan warna biru pada medium yang mengandung laktosa
55Plasmid Polylinkers and Marker Genes for Blue-White screening A vector usually contains a sequence (polylinker) which can recognize several restriction enzymes so that the vector can be used for cloning a variety of DNA samples.Colonies with recombinant plasmids are white, and colonies with nonrecombinant plasmids are blue.Example: pUC19Resistant to ampicillin, has (ampr gene)Contains portion of the lac operon which codes for beta- galactosidase.X-gal is a substrate of beta-galactosidase and turns blue in the presence of functional beta-galactosidase is added to the medium.Insertion of foreign DNA into the polylinker disrupts the lac operon, beta-galactosidase becomes non-functional and the colonies fail to turn blue, but appear white.DNA rekombinan pada plasmid