분자생물학실험 SUBJECT RNA Isolation cDNA synthesis

분자생물학실험 실험계획 주 날짜 실험내용 RNA extraction, RNA quantitation, 1 3/3 Introduction 2 3/10 DNA extraction 3 3/17 Primer design, PCR 4 3/24 Electrophoresis, TA ligation 5 3/31 E. coli transformation 6 4/7 Mini-prep, Electrophoresis, Restriction enzyme 7 4/14 Electrophoresis (Restriction enzyme 결과 확인), Sequence blast 8 4/21 중간고사 9 4/28 RNA extraction, RNA quantitation, cDNA synthesis 10 5/5 휴강 (어린이 날) 11 5/12 RT-PCR, Electrophoresis 12 5/19 Yeast two hybrid (yeast transformation) 13 5/26 Y2H결과 확인, Western blotting (gel running, transfer) 14 6/2 Western blotting (Immunoblotting) 15 분자생물학실험시험 16 6/16 휴강(기말고사 기간)

분자생물학실험

분자생물학실험 RNA Works Northern blot RT-PCR Microarray

분자생물학실험 RNase 에 오염이 되지 않도록 주의!!! 액체질소 사용시 손 조심 (화상) 주의사항 실험대를 청결히 합니다. 장갑착용, 불필요한 말은 삼갑니다. DEPC (RNase inhibitor) 처리한 물 사용. 액체질소 사용시 손 조심 (화상)

분자생물학실험 Tri reagent (Trizol) Materials & Method Combines phenol and guanidine thiocyanate in a mono-phase solution to facilitate the immediate and most effective inhibition of RNase. guanidinium isothiocyanate (powerful protein denaturant) : inactivation of RNases acidic phenol/chloroform : partitioning of RNA into aqueous supernatant for separation

분자생물학실험 Materials & Method 1. Homogenization - Homogenize tissue samples and add 1ml TRI REAGENT. Store the homogenate for 5min. 2. RNA extraction - Add 0.1ml BCP(chloroform) and mix vigorously. - Store sample for 10min at RT. Centrifuge 13,000 rpm for 15min at 4℃(RT) 3. RNA precipitation - Transfer 0.4ml aqueous phase to a clean tube. - Add 0.5ml isopropanol and mix. Store sample for 5min Centrifuge 13,000 rpm for 10min at 4℃(RT) 4. RNA wash - Wash RNA pellet with 1ml 75% ethanol(with DEPC water). Centrifuge 13,000 rpm for 5min. 5. Solubilization - Air dry the RNA pellet. - Dissolve the pellet in 50ul DEPC water and incubate at 55℃ for 10min.

분자생물학실험 Liquid nitrogen 2ml tube Trizol Aqueous phase + isopropanol New tube Aqueous phase + isopropanol 13000rpm chloroform 13000rpm

분자생물학실험 RNA quantification Materials & Method Spectrophotometer [RNA] μg/ml = A260 x 40 x dilution factor Purity = A260 / A280 (1.7~2.0) Electrophoresis rRNA (28S, 18S, 5.8S, 5S), mRNA, tRNA 28S rRNA 18S rRNA Sample : root , leaves

분자생물학실험 Result Spectrophotometer Dilution factor A260 A280 A260 / A280 Purity [RNA] μg/μl Col-0 shr-2 shr-6

분자생물학실험 Result Electrophoresis 결과 1조, 2조 민서 교익 shr-2 shr-6 희연 진희 shr-2 3조, 4조 나연 예원 shr-2 shr-6 선주 규호 shr-2 shr-6

분자생물학실험 Home work Homogenate 에 BCP(Chloroform)를 넣으면 아래 그림과 같이 세 층으로 분리 된다. 산성 조건일때, 각각의 층에 무엇이 존재하는지 쓰시오.

분자생물학실험 다음 실험 예비 레포트 RT-PCR *이번 주 결과 레포트 없습니다.