Introducing 

Prezi AI.

Your new presentation assistant.

Refine, enhance, and tailor your content, source relevant images, and edit visuals quicker than ever before.

Loading…
Transcript

Tammy Wang

MCB

My Intern Experience

Introduction

- MCB group

- 2 1/2 months

- DNA --> Cell --> Protein

Introduction

Molecular Cloning

Molecular

Cloning

Purpose: To insert target DNA into a plasmid

- amplify and manipulate gene of interest

-useful to study a gene

Plasmid- usually a small extra-chromosomal circular piece

Infusion- method

Competent Cells- cells that are permeable to extracellular DNA

- Restriction Enzymes vs Infusion

-also called "In-fusion PCR Cloning"

- newer method

- No additional treatment of the PCR is necessary

PCR

Gel

QIA quick gel

Transformation

-key step to DNA cloning (big step)

-Bacterial transformation is a process where foreign DNA is introduced into the bacteria.

-target DNA--> circular DNA (plasmid)

-The bacteria that accepts the plasmid forms a colony under ampicillan selection

Transformation

Protocol

Purpose: To screen for successful constructs after cloning

size of fragment

Vector

colony sequencing

Colony PCR

Colony PCR

will show up different

Characterization and Purification

Once the previous steps are complete

- Plasmid DNA must be extracted and purified

1) Growth of bacteria

2) Harvesting and Lysis

3) Maxiprep Purification

Maxiprep

Transfection

Mammalian

Expression

-Transfect Plasmid DNA into ExpiCHO cells

- ExpiCHO cells- Chinese Hamster Ovarian

Lipofectamine- increase efficiency of plasmid DNA into ExpiCHO cells

VCD- Viable Cell Density, measured by Trypan Blue Dye Exclusion method

Protocol

Day -1: Split Cells

Split the ExpiCHO cells to ~3x10⁶.

Day 0: Transfect cells

VCD should be 7x10⁶: Viability should be 95-99%.

Day 1: Add ExpiCHO Enhancer and ExpiCHO Feed (Max Titer)

Day 5: Add the second volume of ExpiCHO Feed

Temperature importance

Protocol

1.0 ug total plasmid DNA per mL of culture volume to transfect

0

1

Protein

Pro A: bacteria surface protein

binds to Antibody at the Fc region between CH2 and CH3

- packed as beads in a column

Protein Analysis

Purification

Analysis

SDS-PAGE -Sodium Dodecyl Sulfate-

electrophoresis method- protein separation by mass

= polyacrylamide-based discontinuous gel

- including stacking gel and separating gel

Purification

ProA purification

Analysis

Thank you!

Dr. Zoey Huang

Ms. An Ju and Mr. Michael Hua

Dr. Jiang and Hengenix Biotech Inc.

Acknowledgments

Learn more about creating dynamic, engaging presentations with Prezi