Ex 4: Plasmid purification Objectives Purify plasmid pGEM- T easy with 1kb DNA insert (valL) from the bacteria cells Principles Plasmid purification is an important technique in molecular biology DNA plasmid is purified from bacteria grown in liquid solution There are different methods to purify plasmid, in which the most popular method is to use alkaline solution in the presence of SDS to break bacteria cellular membrane, to denature DNA chromosome and protein, and release plasmid The denatured protein and DNA chromosome are precipitated and are removed by centrifugation while DNA plasmid is in the solution Plasmid will be then precipitated using 100 % isopropanol The concentration and purity of DNA plasmid will be measured in experiment 6, DNA plasmid will be observed on agarose gel in experiment This plasmid will also be used as PCR templates in experiments and and be digested in experiment Exercise 3.1 Materials - Bacteria culture solution which contains vector pGEM- T easy with 1kb DNA insert (valL) - Sol I: 25 mM Tris-HCl pH 8, 50 mM Glucose, 10 mM EDTA pH - Sol II: 0.2 N NaOH, 1% (w/v) SDS - Sol III: M Potassium acetate pH5 - Phenol : Chloroform : Isoamyl (25:24:1) - Isopropanol - Ethanol 70% (cold) - TE buffer: 10mM Tris-HCl pH8 + 1mM EDTA pH 3.2 Protocol - Transfer 1.5 ml overnight bacterial culture into Eppendorf tube, centrifuge at 10,000 rpm for 5’ at ºC Remember to balance the tube before centrifugation - Remove the liquid and collect the cell pellet - Resuspend the cell pellet using 100 µl Sol I vortex Incubate at RT for minutes - Add 200 µl Sol II invert – times until the solution become homogenous, incubate on ice for minutes - Add 150 µl Sol III invert times, incubate on ice for minutes - Centrifuge at 13,000 rpm at 4oC for 15 minutes Collect supernatant, try not to collect cell pellet - Add volume of phenol : chloroform : isoamyl (25:24:1) vortex - Centrifuge at 13,000 rpm, ºC for minutes Collect the upper layer - Add 0.6 volume of isopropanol vortex and incubate at room temperature for – 10 minutes - Centrifuge at 13,000 rpm, ºC for 15 minutes remove the supernatant - Add 0.5 ml of cold, 70% EtOH 70% - Centrifuge at 13,000 rpm, ºC for minutes remove the supernatant, air-dry the pellet - Add 20 - 30 µl TE buffer or water, incubate 37 ºC in 30 minutes, store at º -20 C Write report Explain all the steps in the exercise and function of each of the chemicals in purification of DNA plasmid Are there other methods to purify DNA plasmid? (Vector and DNA fragments are subjected to change in each semester) ... Collect supernatant, try not to collect cell pellet - Add volume of phenol : chloroform : isoamyl (25 :24 :1) vortex - Centrifuge at 13,000 rpm, ºC for minutes Collect the upper layer - Add 0.6... minutes remove the supernatant, air-dry the pellet - Add 20 - 30 µl TE buffer or water, incubate 37 ºC in 30 minutes, store at º -20 C Write report Explain all the steps in the exercise and...- Resuspend the cell pellet using 100 µl Sol I vortex Incubate at RT for minutes - Add 20 0 µl Sol II invert – times until the solution become homogenous, incubate on ice for minutes