Notebook

You know what we did from last winter to autumn! In this Notebook you’ll be able to follow what and when we did it. We documented the experiment protocols and chronological important events, including how we started, how we determined our project, weekly meeting, experiment development, human practice project and many more! Thanks to this Notebook you can travel back in time and see how we managed to get big things done in such a small time frame. Once you get to know the past, you learn to appreciate the present day and the hard work done by CSU_CHINA team.

Protocols

Chemical transformation

Thaw 50µL DH10B competent E. coli cells on ice.
Add 5 µl DNA from a ligation reaction mix or 10-100ng DNA of a plasmid.
Place the mixture on ice for 20-30 minutes.
Heat shock at 42°C for 90 seconds.
Place on ice for 5 minutes.
Pipette 200 µl of room temperature LB media into the mixture.
Incubate at 37°C and 220 rpm for 60 minutes.
Add 50-100 µl of the transformed cells to the selection plate.

Growing overnight cultures

Overnight cultures were prepared under sterile conditions using a Bunsen burner Add 3 mL liquid LB media into test tubes Add 3 μL of appropriate antibiotic（1000x） into the broth Using the pipette tips, pick a single colony and inoculate the cultures by dipping the tip into the LB broth or by adding 50 μL stored cells Put caps on the tubes and incubate overnight at 37°C shaking at 200-250 rpm

PCR From Plasmid DNA Template

In a PCR tube on ice, combine 1-10 ng of plasmid DNA or just 1 µL template, 2 µL of 10 µM forward primer, 2 µL of 10 µM reverse primer to a PCR tube on ice, 25 µL of 2x Phanta Mastermix, and sterile water up to 50 µL. Gently mix the reaction If necessary, collect the liquid to the bottom of the PCR tube by spinning briefly Transfer the PCR tube from ice to a PCR machine preheated to 98°C to begin thermocycling

Preparation of LB Broth

Add 10 g Tryptone, 5 g Yeast Extract and 10 g Sodium Chloride to 1 litre purified water Autoclave

Preparation of LB Agar

Add 10 g Tryptone, 5 g Yeast Extract, 10 g Sodium Chloride and 20 g Agar to 1 litre purified water Autoclave

Ligation

Add vector plasmid, insert DNA, T4 DNA ligase and T4 DNA ligase buffer to the microcentrifuge tube Make reaction up to 10 µL using sterile water Incubate at room temperature for 30 - 60 min

Restriction Digestion

Set up the reaction following the instruction below, depending on whether test digest or assembly digest is being performed. For a Test digest (10 µL): 5 µL Plasmid DNA 0.5 µL Restriction Enzyme 1 0.5 µL Restriction Enzyme 2 1 µL 10× buffer Add sterile water to 10 µL For gene assembly(30 µL): 15 µL Plasmid DNA 1.2 µL Restriction Enzyme 1 1.2 µL Restriction Enzyme 2 3 µL 10X buffer Add sterile water to 30 µL Incubate digestion reaction at 37°C for 30 minutes-1 hour Perform heat deactivation at 80°C for 20 minutes, if not running on a gel at the end of incubation.

What should this page have?

Chronological notes of what your team is doing.
Brief descriptions of daily important events.
Pictures of your progress.
Mention who participated in what task.

Inspiration

You can see what others teams have done to organize their notes:

Team:CSU CHINA/Notebook