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<center>http://farm5.static.flickr.com/4090/5039587233_186da9193f_z.jpg width=30%><br>From the Cambridge 2010 iGEM team</center> | |||
==Overview== | ==Overview== | ||
Revision as of 10:10, 22 November 2010
From the Cambridge 2010 iGEM team
Overview
2 Intro classes: 1 day with two info sessions for people to come and see if they want to take the class or not
- Take place before EHS training
Class 0: for people who haven't taken 7.012: DNA What is synthetic biology
- things that have been done in the past
- potential projects
- Circuits
- what you can do.
What are biobricks
- registry
circuit concepts, circuit design (and/ or gates, NOR gates, etc.)/ feedback
iGEM
What they're doing: bioluminescence
Techniques to teach:
- Lab tour: pipetting, sterile
- (Minipreps)
- We need to make plates, gels
- Labeling
1. Digestion: 1.5 hr 2. Ligation: 1.5 hr, do at the end 3. Gel extract + nanodrop: 20 min prep, 30 min run; 10 min cut, 20 min spin, 5 min nanodrop: 1.5 hr 4. Transformation: 1 hr, 1 hr grow, 20 min plating 5. Picking colonies 20 min--> cell stock--> grow up (us) 6. Miniprep: 1.5 hr, nanodrop 5 min 7. Restriction map
Protocol
Restriction Digest
Recall from your lecture on Biobrick assembly that the cut sites EX-SP surrounding the desired gene allow for restriction enzymes to create sticky ends that can then be used to ligate parts together. In this lab, we will be using the restriction enzymes XbaI, SpeI, and PstI to cut our plasmid DNA.
| Promoter Digestion | Protein Digestion | |||
|---|---|---|---|---|
| Component | Volume (uL) | Component | Volume (uL) | |
| Plasmid | 15 | Plasmid | 15 | |
| Buffer | Buffer | |||
| Water | Water | |||
| BSA | BSA | |||
| SpeI | XbaI | |||
| PstI | PstI | |||
| Total | Total | |||
- Receive 2 tubes of DNA from group leaders. One contains your promoter plasmid, and the other your protein plasmid. They each contain 15uL of DNA in solution.
- You will do all calculations before you begin digestions. First, find the concentration of your DNA. Each tube is different. Then, knowing the concentration of your DNA is in ug/mL, calculate the ug of DNA in your tube.
- You will use 10 units of enzyme per ug of DNA in your tube. Use the table below to calculate how many uL of enzyme you will use to digest your DNA.
| Enzyme | Concentration (units/mL) |
|---|---|
| EcoRI-HF | 20,000 |
| XbaI | 20,000 |
| SpeI | 10,000 |
| PstI | 20,000 |
