Go. : : Spectrum of Innovation CHROMOUS BIOTECH Pvt, Lp. RESTRICTION DIGESTION TEACHING KIT: (3 enzymes) INTRODUCTION: * Restriction enzymes are defense mechanism for the bacteria against viral attack Restriction enzymes are the molecular scis it * Till Date, more than 200 different microbial sources * Out of four different categories of restriction modification (RM) system known, most used Restriction enzymes belong to Type II Class, Restrieven enzymes are known t9 Br sane ither blunt or sticky ends upon digestion. ‘Sma I genenaee blunt ends, Eco RI generates sticky ends with 5"-overhang while Pst 1 generates sticky ends with 3°- overhangs. 7 * Two independent enzymes generating overhangs that can ligate with each other are called 'soschizomers © fhete are Restriction enzymes that recognize and cut 4 bases (4 base cutters) and there are 16-base cutters as well, * Enzyme units are estimated as the amount of enzyme required to completely digest fonditions. Optimal temperature condition for restriction enzyme activity ranges between 25°C (Sma I) to 65°C (Taq 1) * Vendors supply Restriction enzymes containing storage buffer with 50% glycerol, at 20°C. Reaction assay buffers specific for the enzyme are provided at 10X concentration, * Most Resiriction enzymes are stable for Iyear or more if stored properly at -20°C. KIT DESCRIPTION: The kit demonstrates restriction digestion of @ plasmid DNA, a pUCI8 derivative (clone), using 3 different Restriction Enzymes, Eco RI, Hind III and Bam HI. The restriction loaded onto a 1% agarose gel and stained with ethidium bromide, fragments/ bands obtained are viewed under UV light. PRECAUTIONS: {Read the instruction manual before starting the experiment. * ihe staining dye contains Ethidium Bromide. This is known to be carcinogenic and hence should be handled with extreme care. Use eleven while handling, the staining dye and the Agarose gel All the vials provided are to be spun before use (short spin 10000 tpm for | min) Enzymes are sensitive to temperature. Always keep the enzymes at -20° c Switch on the dry bath, before Starting and set the temperature to 37°Cneg amet CHROMOUS BIOTECH PVT. LTD. ‘The reagents provided for preparing and running Agarose gels are enough for casting and running 5 nos. of 1% gels of 50 ml volume each, Minimum two experiments are to be carried out simultaneously and samples are loaded on to same gel KIT COMPONEN’ The components should be stored as suggested for best results, Use the kit within Lyear of arrival. The materials provided in the kit include: Component Quantity Storage Modified pUC18 plasmid DNA (100ng/ul) | 600 yl -20°C Eco RI (SUA) S 10 pl 20°C Bam HI (SU/ul) . 10 pl 20°C Hind TH (5U/ul) 10 pl 20°C ‘Assay buffer (10X) 100 pl =20"C ‘500 bp Ladder (Ready-to-load) 200 il (for 10 loadings) _| -20°C ‘Agarose 258 RT 6X GLB 120 pl RT 50X TAE 25 mb RT Staining dye 25 pl RT 1.5 ml vials 30 nos, RT Water 7a 1.5 mi 7 MATERIALS REQUIRED (Not included in the kit): Dry bath, crushed ice, tips, micropipette PROCEDURE: Note: Minimum two experiments are to be carried out simultancously. Prepare 1% agarose gel before setting up the restriction digestion reactions, Allow the agarose to set for Ihr at room temperature, Restriction digestion reaction: * Modified pUC18 plasmid DNA is digested with three restriction enzymes ‘© Three separate reactions have to be set up * Take out the plasmid DNA; assay buffer kept in -20° C and put them on crushed ice. Allow the components to get thawed on ice. * Prepare the reaction mix as given below: EcoRI reaction mixture: Plasmid DNA 15.041 Assay buffer (10X) 3.0 ul EcoRI enzyme (SU/u!) 1.0 ul Water 11.0 ul Total 30.0 pl MTKO1 (10 reactions) Page 2 of 4CHROMOUS BIOTECH PVT. LTD, Spectrum of Innovation Bam HI reaction mixture: Plasmid DNA 15,0 ul Assay buffer (10X) 3.0 ul Bam HI enzyme (5U/l) 1.0 yl Water 11.0 pl Total 30.0 ul Hind III reaction mixture: Plasmid DNA. 15.0 ut Assay buffer (10X) 3.0 yl Hind Ul enzyme (SU/l) 10 nL Water 11.0 pl a Total 30.0 ul © Add all the components for the mi spin at 10000 rpm for 20 sec ° Incubate the mix at 37°C dry bath for 1 hour ix and tap it 2-3 times for mixing. Give a short Agarose gel electrophoresis: * Tispare 1 % Agarose gel before setting up the restriction digestion reaction Do not disturb the gel tll it solidifies at room temperature for about an howe * Add 200 ml 1X TAE to the gel and add 5 lof staining dye to the buffer in the gel tank. Mix thoroughly by tilting the gel tank 5-6 times ¢ Prorun the gel for 5 min before loading the samples, * After the restriction digestion reaction, add 3 yl of 6X GLB to each of the reaction mix in vials ot the entire volume ofthe digested samples on the Agarose gel, © Load 15 ul of undigested plasmid DNA (mixed with 3 ul of 6X GLB) and 20 yl of 300 bp ladder (ready-to-load) along with the digested samples Electrophorese the samples for 1 hr at 100 V Visualize the gel under UV transilluminator. OBSERVATION: * Note down the size of the fr: with that of the ladder. {Observe the banding pattern obtained by three restriction enzymes * Molecular weight of the 500 bp ladder starts form SOObp, 1kb, 1 5kb, 2kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb and 5,kb. ° Undigested plasmid if présent in digested sai along with digested bands of expected size. ‘agments as seen on Agarose gel under UV comparing imples would show up as linearized band ‘MTKOI (10 reactions) Page 3 of 4CHROMOUS BIOTECH PVT. LT Lane description: 500 bp ladder Undigested Plasmid Bam HI digestion Eco RI digestion Hind II digestion vRepe Restriction Digestion pattern of Modified pUCI8 plasmid INTERPRETATION: * From the banding pattem, one can note that each restriction enzyme has specific recognition site and cuts onlv at specific position, The recognition site is unique for a particular enzyme * Modified pUC provided has only one recogu.tion site fr Bam HI, hence digestion with bum HI resulted in single cut plasmid, a linear uand can be seen near 5 kb. There .. three Eco RI recogmuion sites in the modified plasmid. Hence the restriction digestion with Eco RI results in three bands of size » kb, 1400 bp and 1100 bp. ‘* Hind III had two recognition sites in the modified pUC plasmid. Hence the restriction digestion with Hind II! results in two bands of size 3700 bp and 1800 bp. Q&A 1, What is star activity? Restriction enzymes, when added in excess units and incubated for extra long hours, are known to generate nonspecific cuts, known as star activity. Most enzymes demonstrate star activity. Other factors like cofactor concentration, substrate DNA quality, etc. are also known to enhance star activity by Restriction enzymes. 2. Should we use excess units of enzymes? Why? Usually, for complete restriction digestion of substrate DNA, (3-5) fold excess enzyme is used. Initially 2-fold excess enzyme is used for 45 to 60 mins followed by further addition of I-fold of enzyme. Digested DNA should be loaded on agarose gel for testing. If uncut DNA seen on gel, more enzyme can be added by increasing the reaction volume proportionately with excess 10X assay butler added. 3. Why there are such stringencies of reaction buffers? The Restriction enzymes are isolated from different microbes growing under different biological conditions, Thus, individual Restriction enzymes have independent activity conditions for optimal performance, viz., temperature, pH, lonic concentration, cofactors, etc. 4. What do you sce when native plasmids are digested and run on agarose gel? Good quality purified Plasmid DNA isolates consist mostly of CCC (covalently closed circular) DNA. with small fractions of contaminating nicked and linear forms. Upon completion of restriction digestion, the plasmid appears as linear DNA running slower/heavier than the CCC form. Thus, plasmids with 3 forms (bands) would show up as a single form (band) upon restriction digestion. MTKOI (10 reactions) Page 4 of 4