Outline for Intro to Standard Assembly
PREPARATION - make lots of copies of the parts you want to work with (and save some for later!) SEE iGEM 07 Parts Kit docs, BioBricks Construction Tutorial
1. resuspend the parts of interest in their wells with 15 uL of dH20
- How do I find the right well for part BBa_XXXX?
- What do I do with the foil top?
- Where should I store my plates?
- Hey, I can't see anything in the wells...
2. take 1uL and transform into competent cells - 1 hour
- What kind of transformation - chemical or electroporation? What if I need more than 1uL?
- What kind of cells do I need (top10?)
- What should I do with the other 14uL of part dna?
3. Plate the cells on selective media to isolate a clonal population - 18 hours / overnight
- How do I make plates?
- How do I make antibiotic stocks?
- What kind of antibiotic do I need?
- What's the big deal with plating & "clonal populations" anyway? And "selection" & "markers"?
- How long should I grow up the plates, and at what temperature?
- Oh Noez! The plates stayed in for an extra day / were 30 degrees too hot! what now?
4. Pick a single colony and inoculate in selective broth - 18 hours / overnight
- How do I make broth?
- How much broth should I use? (5 ml)
- What's the difference between Luria Broth and ???
5. Use some of the liquid culture to make a glycerol, and use the rest to do a miniprep to isolate the plasmid that contains your biobrick of interest - 1 hour
- How do I make a glycerol? Where do I store it? How do I use it?
- What's a miniprep? How does it work? Should I spec it?
STANDARD ASSEMBLY - isolate part_A from its plasmid and insert it before or after part_B in part_B's plasmid SEE Assembly:Standard Assembly
6. Do a restriction digest of the plasmid containing part_A to cut part_A out and prepare the correct pattern of sticky ends.
- How does the BioBrick standard work?
- What are restriction enzymes, why is it called a digest?
- Where do I get enzymes, where do I store them?
7. Gel purify the cut fragments of the part_A plasmid
- How do I make a gel? What do I need to take into consideration?
- Do I need to do anything to the digestion product to prepare it for the gel?
- How does gel purification work? How do I get the DNA out of the gel?
8. Do a restriction digest of part_B's plasmid to prepare the pattern of sticky ends compatible with those of part_A
- no gel purification needed... but what about getting rid of the restriction enzymes?
9. Ligate part_A with the linearized plasmid containing part_B
- Are ligation enzymes and restriction enzymes like matter and antimatter?
- how can I be sure everything ligates correctly? I do I prevent incorrect / unintended ligation products?
10. Transform the ligation product into competent cells
- Anything special about this transformation?
11. Plate the cells
- how can you screen for a successful composition?
- what sources of error could confound a particular screen? (i.e. incomplete digestion)
12. Make a glycerol stock
13. goto step 1.