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.