Loading presentation...

Present Remotely

Send the link below via email or IM

Copy

Present to your audience

Start remote presentation

  • Invited audience members will follow you as you navigate and present
  • People invited to a presentation do not need a Prezi account
  • This link expires 10 minutes after you close the presentation
  • A maximum of 30 users can follow your presentation
  • Learn more about this feature in our knowledge base article

Do you really want to delete this prezi?

Neither you, nor the coeditors you shared it with will be able to recover it again.

DeleteCancel

Make your likes visible on Facebook?

Connect your Facebook account to Prezi and let your likes appear on your timeline.
You can change this under Settings & Account at any time.

No, thanks

유전공학기말

No description
by

보성 김

on 7 December 2012

Comments (0)

Please log in to add your comment.

Report abuse

Transcript of 유전공학기말

1. Cell preparation & Permeabilization





Fixation : Use PFA to fix the cell.

Permeabilization : Use Triton X-100 to increase cell membrane and nuclear membrane permeability.
Monday Team
2010140115 Jin YunJung
2010140123 Kim BoSung
2010140124 Cho SeokHee Biotechnology
& Experiment Oct-4 is a marker for pluripotent human stem cells to be expressed in embryonal carcinoma cells, embryonic stem cells and embryonic germ cells. Tera-2 Cell
: Tera-2 embryonal carcinoma cells, obtained from a nonseminomatous testicular germ cell tumor. Tera-2 cells express the Oct-4 gene only in the undifferentiated state. AF Cell
: Human Amniotic Fluid Cell can express Oct-4 in the nucleus. It can be divided into two category, Oct-4-negative cell and Oct-4-positive cell. AF Cell & Tera-2 Cell Ⅰ. Genomic DNA PCR Ⅰ. Genomic PCR
Ⅱ. RNA RT PCR
Ⅲ. Immunocytochemistry Contents 1.Genomic DNA extraction –O.D value
2.Genomic DNA PCR –Gel Electrophoresis Genomic DNA PCR
Results & Discussion SH

AF
O.D=17
260/230=0.47
260/280=2.04

Tera-2
O.D=76
260/230=0.41
260/280=1.85 BS

AF
O.D= 22
260/230= 0.64
260/280= 1.78

Tera-2
O.D= 60
260/230= 0.36
260/280= 1.99 YJ

AF
O.D=29
260/230=0.25
260/280=1.99

Tera-2
O.D=103
260/230=0.39
260/280=1.88 – O.D value Genomic DNA PCR Results & Discussion BS YJ – hOct4 Primer(10pM) add for PCR SH 121029 Genomic DNA hOct4 –YJ, BS, SH - Gel electrophoresis – Genomic DNA hOct4 PCR – 62ºC, 30 Cycle 석희 보성 윤정 2. Genomic DNA PCR Gel Electrophoresis 150bp C AF TERA C AF TERA C AF TERA YJ BS SH Ⅱ. RT PCR 1. RNA Prep –O.D value
2. RNA RT PCR
3. GAPDH PCR–Gel Electrophoresis
4. hOct4 PCR –Gel Electrophoresis RT PCR Results & Discussion BS

AF
O.D=100
260/230=1.35
260/280=2.00

Tera-2
O.D=384
260/230=1.96
260/280=2.07 SH

AF
O.D=114
260/230=1.81
260/280=2.01

Tera-2
O.D=640
260/230=2.18
260/280=1.84 YJ

AF
O.D=149
260/230=1.79
260/280=2.06

Tera-2
O.D=497
260/230=1.98
260/280=2.03 – O.D value RNA RT PCR Results & Discussion –RT PCR -42 ℃, 94 ℃, 24 Cycle 3. GAPDH PCR
– Gel electrophoresis C AF Tera C AF Tera C AF Tera – Gel electrophoresis – GAPDH PCR -62 ℃, 24 Cycle
121109 GAPDH- 1st YJ, BS, SH C AF Tera C AF Tera C AF Tera AF Tera 121112 GAPDH- 2nd YJ, BS BS YJ – GAPDH PCR -62 ℃, 24 Cycle SH 1. Do not dry ethanol sufficiently.
2. Skip over pipetting procedure of the RNA.
3. Do not put the GAPDH primer into PCR mix. YJ BS SH C AF Tera C AF Tera C AF Tera – Gel electrophoresis – PCR 62 ℃, 35 Cycle

121112 hOct4 – 1st YJ, BS, SH 4. hOct4 PCR
–Gel electrophoresis
AF
O.D=489
260/230=2.10
260/280=2.03

Tera-2
O.D=640
260/230=2.18
260/280=1.84 – O.D value Second RNA Prep -O.D value RNA RT PCR Results & Discussion – GAPDH PCR -62 ℃, 24 Cycle Second GAPDH PCR – Gel electrophoresis C A① A② T C A① A② A③ T Tera-2 : 1 λ Tera-2 : 0.8 λ 121113 GAPDH- 2nd SH AF Tera
121116 hOct4 – 2nd SH YJ BS SH Tera AF C Tera AF C Tera AF C – Gel electrophoresis – PCR 62 ℃, 24 Cycle

121116 hOct4 – YJ, BS, SH YJ BS SH YJ BS Ⅲ. Immunocytochemistry 1. Cell preparation & Permeabilization
2. Adding 1st Antibody (hOct4 Ab)
3. Adding 2nd Antibody (CY3 Ab)
4. DAPI staining Immunocytochemistry
Results & Discussion 1. Cell preparation & Permeabilization
- Cell culture, Fixation, Permeabilization

2. Antibody treatment
- 1st Ab : hOct4 Antibody
2ndAb : CY3 Antibody
3rd Ab : DAPI Antibody

3. Observation
- with Fluorescence microscope Results & Discussion Results 2. Antibody treatment & Observation
1) YJ ’s fluorescence microscope

AF DAPI Tera-2 DAPI Results 2. Antibody treatment & Observation
1) YJ ’s fluorescence microscope

Tera-2 CY3 Tera-2 CY3 & DAPI MERGE Results 2. Antibody treatment & Observation
2) BS ’s fluorescence microscope

AF DAPI Tera-2 DAPI Results 2. Antibody treatment & Observation
2) BS ’s fluorescence microscope

Tera-2 CY3 Tera-2 CY3 & DAPI MERGE Results 2. Antibody treatment & Observation
3) SH ’s fluorescence microscope

AF DAPI Tera-2 DAPI Results 2. Antibody treatment & Observation
3) SH ’s fluorescence microscope

Tera-2 CY3 Tera-2 CY3 & DAPI MERGE Results Thank You
for Listening :) On this semester 1. DNA Cloning

2. Genomic Oct-4 DNA PCR

3. Oct-4 DNA PCR from cDNA

4. Observation Oct-4 protein in cell RESULT 1. CY3 DYE - 550 nm Absorbance
570 nm Emission - red color 2. DAPI DYE - 365nm Absorbance
450nm Emission- blue color 1.Genomic DNA extraction Q&A
Full transcript