Section II. DNA isolation and analysis 1. Plasmid prep 2. Nucleic acids-RNA, DNA 3. Agarose Gel 4. BioEdit - 소개 Working with DNA- DNA cloning Section III. Restriction endonucleases 1. Nomenclature, unit definition (linear vs suprtcolied), Recognition seq & frequency. Isoschemers 2. Reaction- buffers, incubation temp, stopping, double digestions, common inhibitors, 3. Cleavage close to the end (PCR products) 4. Star activity 5. Effects of Methylations- Dam & Dcm methylation, CpG methylation 참고문헌 - Molecular Cloning Section IV. DNA modyfying enzymes 1. Ligases- DNA ligases, RNA ligases 2. Kinase & phosphatase 3. DNA polymerase- Klenow enzyme, T4 DNA polymerase, PCR enzymes 4. Nucleases- S1 nuclease, Mung Bean Nuclease Section I. Host & vector system 1. E.coli strains - genotypes 2. Host-Vector systems

Section I. Host & vector system 1. E.coli strains - genotypes 2. Host-Vector systems Section II. DNA isolation and analysis 1. Plasmid prep 2. Nucleic acids-RNA, DNA 3. Agarose Gel 4. BioEdit - 소개 Section II. DNA isolation and analysis Alkaline lysis- Glucose/Tirs/EDTA SDS/NaOH Potassium Acetate Phenol extraction/Ethanol precipitation Phenol/Chloroform/isoamyl alcohol Ethanol precipitation Ethanol wash Dry

Nucleic acid quantitation Resolve in Sterilized Distilled water, 10mM Tris-1mM EDTA, 10mM Tris-1mM EDTA-100mM NaCl Storage 4’C vs Freezer

Agarose Gel Electrophoresis 1. agarose gel %? 2. TBE vs TAE buffer Tris-base/boric acid/EDTA Tris-base/Acetic acid/EDTA 3. Loading buffer: Tris-EDTA, glycerol(or sucrose), dye 4. How much DNA/well? 3ul~ 수십 ul/well; 수십 ng/band

실험 1- Plasmid isolation with a kit high & middle copy number plasmid pUC18 isolation 숙제 1.: E.coli EPI300 의 genotype 과 그 유전자들의 의미를 정리하여 제출. 숙제 2: 정제한 pUC19 DNA solution 의 흡광도가 다음과 같을 때 DNA 의 concentration (ug/ul) 와 pUC19 의 molarity (XX uM) 을 구하세요 흡광도 No dilution-- OD260=3.2 1/50 dilution-- OD260=0.8 숙제 3- pUC18(or19) 의 DNA sequence 를 internet 에서 받아 BioEdit 으로 Restriction map, reading frame map 그려 제출할 것