LB media

Final concentrations:

• 1% (w/v) tryptone
• 0.5% (w/v) yeast extract
• 86 mM NaCl

Per liter:

• 10g Tryptone
• 5g Yeast extract
• 5g NaCl




• 15g Agar -Optional-
• Adjust pH to 7.5 with NaOH 1M

SOB media

Final concentrations:

• 0.5% (w/v) yeast extract
• 2% (w/v) tryptone
• 10 mM NaCl
• 2.5 mM KCl
• 20 mM MgSO4

Per liter:

• 5 g yeast extract
• 20 g tryptone
• 0.584 g NaCl
• 0.186 g KCl
• 2.4 g MgSO4




• 15g Agar -Optional-
• Adjust pH to 7.5 with NaOH 1M

5X Gibson Assembly Isothermal Buffer

Final Concentrations:

• 25% PEG MW 8000
• 500mM Tris HCl pH 7.5
• 50mM CaCl
• 50mM DTT
• 1mM dATP
• 1mM dTTP
• 1mM dGTP
• 1mM dCTP
• 5mM NAD+
• H20 nuclease free

For a 2 mL stock (equivalent to 20 100uL 5X isothermal buffers)

• 0.5g PEG MW 8000
• 1.5mL Tris-HCl pH 7.5 1M

Swivel for 30 minutes in a orbital oscillator for PEG to dissolve fully

• 50uL CaCl 2M
• 100uL DTT 1M
• 20uL dATP 100mM
• 20uL dTTP 100mM
• 20uL dGTP 100mM
• 20uL dCTP 100mM
• 200uL NAD+ 200mM
• H20 nuclease free up to 2mL

Alicuot into 100uL stocks

Store at -80°C

1.33X Gibson Assembly Master Mix

For a 375ul 1.33X Gibson Assembly Master Mix

• 100uL 5X Gibson Assembly Isothermal Buffer
• 6.25uL Phusion Polymerase 2 U/uL (cat N° F-350S) from Thermo Scientific
• 2uL Epicentre T5 Exonuclease 1U/uL (cat N° T5E4111K) from Illumina

It is important to dilute the T5 Exonuclease stock from 10U/uL to 1U/ul to measure the volume correctly

• 50uL Taq DNA Ligase 2000 U/uL (cat N° M208S) from NEB

Gibson Assembly