Protocols

Making LB media

Dissolve 12.5g of LuriaBroth in 500mL MilliQ H2O and autoclave

Making LB-Agar

Dissolve 12.5g LuriaBroth in 500mL MIlliQ H2O,add 7.5g Agar.
Before use: Heat it inb the microwave and let it cool down to 50 degree celsius before adding Antibiotics.

Antibiotics stock solutions

Ampicillin (amp): 100mg/mL (1000X)
Dissolve 1g Ampicillin in 10mL sterile H2O.Aliquot and store at -20 degree celsius.

Kanamycin (Kan): 50mg/mL (1000X)
Dissolve 0.5g Kanamycin in 10mL sterile H2O.Aliquot and store at -20 degree celsius.

Chloramphenicol(cam): 34mg/mL (1000X)
Dissolve 0.34g Chloramphenicol in 10mL sterile H2O. Aliquot and store at -20 degree celsius.

All stock solutions have to be diluted 1:1000 times when used for Cultures/Plates

Glycerol 20%

Mix 20mL of Glycerole 100% and 80mL of MilliQ H2O, autoclave

Restriction enzyme digest

50uL reaction Volume:
2ug of plasmid (max 45uL for low concentration minipreps)
5uL NEB buffer (which buffer for which enzzyme can be looked up on the NEB website)
add ddH2O up to 50uL
0.5/1uL of restriction enzyme (20000U/mL/10000U/mL)
Keep enzymes always on cooling block and don't take them out for too long.
Mix through pipetting

1h at 37 degree celsius
Heat inactivation : 20min at 65 degree celsius(not always necessary)

Dephosphorylation of backbones only (reduces plasmid slef ligation)
Add 1uL of Calf-intestine-phosphatase)
1h at 37 degree celsius
20 minutes heatinactivation at 65 degree celsius.

Add 10uL of loading buffer (6X)
Analysis on gel

Figure 1. iGEM suffix insertion

Figure 2. iGEM 3A Assembly