Electrophoresis Flashcards
What is agarose?
A linear carbohydrate polymer extracted from seaweed
To imrove yield sample
- Use more starting material
- Use fresh starting material
- Extend the precipitation period 20 C (to hours/days)
What is Temple’s favourite color?
Emerald green
To improve yield sample?
- Use more starting material
- Use fresh starting material
- Extend the precipitation period at 20*C ( to hours?days )
What is gel electrophoresis
Is a technique used to saperate DNA fragments according to size.
Equipmenents used
Gel tray. Gel tank,
Electrodes ( leading to a power pack )
Visualizing fragments
- Digital camera
- Emission filter
- Excitation light
- Excitation filter
- DAG - DNA Agarose Gel
Excitation light
- Blue LED
- Cyan LED
- Black light
Which system is used to record gel?
Geldoc system
Visual Procedure
- DNA sample loading
- Application of electrical field
- DNA separation
- UV transillumination + Documentation
Why are fragments arranged in sizes ?
Shorter strands of DNA moves more quicker through the gel than longer strands
Polyacrylamide gel electrophoresis units?
- kDa = Protein
- bp, Mb or Kb = DNA or RNA
DNA INTEGRITY: by gel electrophoresis
High MW gDNA & Protein
- Increased amount of Proteinase K for protein contamination
- Increased period of precipitation step to increase yield.
DNA INTEGRITY: by gel electrophoresis
Degraded gDNA
- Work at 4*C throughout protocol
- Minimize shearing by being “ more gentle “
DNA INTEGRITY: by gel electrophoresis
RNA
- Add an RNAse to remove RNA
- increased extraction buffer pH (to favour DNA over RNA)
- Add Lithium Chloride in ethanol precipitation step
Why add Lithium Chloride in ethanol precipitation step?
It precipitates RNA but not Proteins or DNA.
Why does DNA absorb UV light
Due to heterocyclic rings of the nucleotides
