1. 분자생물학실험
SUBJECT
RNA Isolation
cDNA synthesis

2. 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)

4. 분자생물학실험

5. 분자생물학실험
RNA Works
Northern blot
RT-PCR
Microarray

6. 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)

7. 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

8. 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.

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

10. 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

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

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

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

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