What colonies are considered as a successful transformant, blue colonies, white colonies or both?
5.4.1 Screening by blue/white colour
- What colonies are considered as a successful transformant, , white colonies or both?
- Step 9: Which part contains the plasmid DNA (of interest), clear lysate or white precipitate?
- Step 10: What’s the purpose of adding 100% ethanol?
- Step 12: What’s the purpose of adding 70% ethanol?
- Step 16: Why do we airdry the DNA sample before resuspending it in water?
5.4.3 Agarose gel electrophoresis
- What’s the purpose of having a blue uncut sample loaded onto the gel?
- What’s the purpose of having a blue EcoRI-HF digest loaded onto the gel?
- What’s the purpose of having a white uncut loaded onto the gel?
- Step 4 Why do we centrifuge the tubes after restriction enzyme digest?
- What are the expected band size in lane 1 to 6 for complete and incomplete digests (assuming white colonies are positive, and they contain the correct insert)?
| Lane 1
Blue colony Uncut |
Lane 2
Blue colony EcoRI-HF |
Lane 3
White colony 1 Uncut |
Lane 4-6
White colony 1-3 EcoRI-HF |
|
| Expected
Band size (complete digest) |
||||
| Expected
Band size (incomplete digest) |
5.4.4 Expression of GFP in transformed colonies
- Why did we preselect 3 white colonies under UV last week considering we are going to check their expression under the microscope this week?
- Step 5: What would you conclude if you have a white colony and it fluoresces under the microscope?
What colonies are considered as a successful transformant, blue colonies, white colonies or both?
