BioBricks construction tutorial: Difference between revisions
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Barry Canton (talk | contribs) (→Steps) |
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Assumed starting point is from glycerol stocks of insert and vector. | Assumed starting point is from glycerol stocks of insert and vector. | ||
#[[Streak plates]] of | #[[Streak plates]] of insert and vector from glycerol stocks. | ||
#[[Bacterial cell culture|Grow 5ml Cultures]] of each from a | #[[Bacterial cell culture|Grow 5ml Cultures]] of each from a single colony | ||
#[[Miniprep]] | #[[Miniprep]] plasmid DNA from cultures | ||
#[[Restriction digest]] | #[[Restriction digest]] insert and vector with appropriate enzymes | ||
#[[DNA Gel extraction|Extract]] | #[[DNA Gel extraction|Extract]] vector and insert from gel | ||
#[[DNA Ligation|Ligate]] | #[[DNA Ligation|Ligate]] insert and vector | ||
#[[Electroporation|Transform]] | #[[Electroporation|Transform]] ligation product into competent cells | ||
#*Can use [[Electroporation|electroporation]] or [[Chemically competent cells|chemical transformation]] | #*Can use [[Electroporation|electroporation]] or [[Chemically competent cells|chemical transformation]] | ||
#Screen | #Screen colonies for correct plasmid by PCR | ||
#[[Miniprep]] DNA from | #[[Miniprep]] DNA from chosen colony | ||
#Make | #Make glycerol and sequence DNA | ||
Revision as of 15:13, 25 August 2005
Contacts
Barry Canton, Jason Kelly, Will Bosworth
Aim
Describe the steps of a standard BioBricks assembly for two parts and include images of expected results (e.g., gels, seq files, etc). This will provide a walkthrough for someone new to BioBricks construction to practice with parts that are known together well.
Steps
Assumed starting point is from glycerol stocks of insert and vector.
- Streak plates of insert and vector from glycerol stocks.
- Grow 5ml Cultures of each from a single colony
- Miniprep plasmid DNA from cultures
- Restriction digest insert and vector with appropriate enzymes
- Extract vector and insert from gel
- Ligate insert and vector
- Transform ligation product into competent cells
- Can use electroporation or chemical transformation
- Screen colonies for correct plasmid by PCR
- Miniprep DNA from chosen colony
- Make glycerol and sequence DNA
