BioBricks construction tutorial: Difference between revisions
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==[[Electroporation|Transform]] ligation product into competent cells== | ==[[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 colonies for correct plasmid by PCR== | ==Screen colonies for correct plasmid by [[Colony PCR]]== | ||
<show picture of plate under FL light here, to show the sucessful transformants glowing.> Should probably point out that it's usually not this easy to screen. We can also tell them to pick a couple that glow and a couple that don't to confirm that the colony pcr does a good job at predicting a successful tranformation. | <show picture of plate under FL light here, to show the sucessful transformants glowing.> Should probably point out that it's usually not this easy to screen. We can also tell them to pick a couple that glow and a couple that don't to confirm that the colony pcr does a good job at predicting a successful tranformation. | ||
==[[Miniprep]] DNA from chosen colony== | ==[[Miniprep]] DNA from chosen colony== | ||
==Make glycerol and sequence DNA== | ==Make glycerol and sequence DNA== | ||
Revision as of 15:30, 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
- Antibiotic Used:
- Incubation Time:
- Image of Plate after growth
Grow 5ml Cultures of each from a single colony
- Cellular chassis: ?
- Growth media: ?
- Antibiotic: ?
- Growth Temperature: ?C
- Spin speed: ?rpm
- Incubation Time: ?hrs
- Final OD600:
Miniprep plasmid DNA from cultures
- Prep concentration vector: ?ng/μl (?μl elution volume)
- Prep concentration insert: ?ng/μl (?μl elution volume)
Restriction digest insert and vector with appropriate enzymes
- Vector restriction enzymes used:
- Insert restriction enzymes used:
- Incubation time:
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 Colony PCR
<show picture of plate under FL light here, to show the sucessful transformants glowing.> Should probably point out that it's usually not this easy to screen. We can also tell them to pick a couple that glow and a couple that don't to confirm that the colony pcr does a good job at predicting a successful tranformation.
