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Team:UC Chile2/General Protocols
From 2012.igem.org
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Revision as of 03:22, 12 September 2012
Contents |
Growth Media
LB media
- 1% (w/v) tryptone
- 0.5% (w/v) yeast extract
- 86 mM NaCl
- 10g Tryptone
- 5g Yeast extract
- 5g NaCl
- 15g Agar -Optional-
- Adjust pH to 7.5 with NaOH 1M
Final concentrations:
Per liter:
SOB media
- 0.5% (w/v) yeast extract
- 2% (w/v) tryptone
- 10 mM NaCl
- 2.5 mM KCl
- 20 mM MgSO4
- 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
Final concentrations:
Per liter:
Buffers
5X Gibson Assembly Isothermal Buffer
- 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
- 0.5g PEG MW 8000
- 1.5mL Tris-HCl pH 7.5 1M
- 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
Final Concentrations:
For a 2 mL stock (equivalent to 20 100uL 5X isothermal buffers)
Swivel for 30 minutes in a orbital oscillator for PEG to dissolve fully
Alicuot into 100uL stocks
Store at -80°C
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
- 50uL Taq DNA Ligase 2000 U/uL (cat N° M208S) from NEB
For a 375ul 1.33X Gibson Assembly Master Mix
It is important to dilute the T5 Exonuclease stock from 10U/uL to 1U/ul to measure the volume correctly
