Transfection Flashcards
(14 cards)
Why transfect?
- Shorter time to results
- drug discovery/efficacy
- 1-4 days as opposed to 3 months
- Option for Temporary Alterations
- transient protein expression
- temporary expression knockdown
- Can alter cells without altering the germline
How do cells protect Against Foreign DNA?
- Nucleic Acids are negatively charged
- cells are designed to prevent entry
- defense against bacteria, virus, etc.
- size can also be an issue
- plasmids can get upwards of 15 kb
- anything larger than 10 kb reduces efficiency
Whats happening?
Nucleic Acids (DNA/RNA) are artificially introduced into cells
General Workflow of Transfection
- Transfection reagent is added to DNA/RNA
- incubate for 20 minutes
- transfection complex is added to cells
- assay cells 24-72 hours post transfection
What considerations should be made when picking transfection reagents?
- toxicity
- how much does this method harm your cells?
- efficiency
- can this method efficiently transfect your cell type?
- Downstream Applications of Cells
- is this method compatible with methods that will be used to analyze the cells after transfection
- Cost
Transfection Methods
- Lipid based
- Ion Based
- Electroporation
- Gene gun - usually for plants
Transfection Methods: Lipid Based
most common - lipofectamine 2000
positively charged lipids
Transfection Methods: Ion Based
Positively Charged ions
Cheaper option
Most common: Calcium Phosphate
Transfection Methods: Electroporation
Positive Ions punch holes in the cell membrane
Cell characteristics that make transfection difficult:
Primary Cells
Non-dividing cells
Technical Challenges: Confluence
how tightly packed is your plate
Transfection Methods: Growth rate
transfections need dividing cells
Media
serum inhibits transfections
need to use OptiMem or serum free media
Downstream: Efficacy Tests
- Microscopy
- fluorescent proteins (reporter protein)
- Enzymatic Assay
- Luciferase assay
- DNA → PCR
- RNA → RT PCR
- Protein → Western Blot
