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- MCB group
- 2 1/2 months
- DNA --> Cell --> Protein
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
QIA quick gel
-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
Purpose: To screen for successful constructs after cloning
size of fragment
Vector
colony sequencing
Colony PCR
will show up different
Once the previous steps are complete
- Plasmid DNA must be extracted and purified
1) Growth of bacteria
2) Harvesting and Lysis
3) Maxiprep Purification
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
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
1.0 ug total plasmid DNA per mL of culture volume to transfect
0
1
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
Dr. Zoey Huang
Ms. An Ju and Mr. Michael Hua
Dr. Jiang and Hengenix Biotech Inc.
Acknowledgments