Editing Gibson Assembly (section)

== Protocol 4 ==

MATERIALS 

beta-Nicotinamide adenine dinucleotide (NAD+) - 0.2 ml, New England Biolabs Catalog #B9007S 

Q5 High-Fidelity DNA Polymerase - 100 units , New England BiolabsCatalog #M0491S 

Taq DNA Ligase - 2,000 units, New England BiolabsCatalog #M0208S 

T5 Exonuclease - 5,000 units, New England BiolabsCatalog #M0363L 

PEG-8000&nbsp; 

DTT (Dithiothreitol) (> 99% pure) Protease free,&nbsp; Gold Biotechnology Catalog #DTT 

Deoxynucleotide Solution Set - 25 umol of each, New England Biolabs Catalog #N0446S

Recipe for 4 mL:

{|
|-
| colspan="1" rowspan="1" | Component
| colspan="1" rowspan="1" | Molarity / Concentration
| colspan="1" rowspan="1" | Amount
| colspan="1" rowspan="1" | Final concentration
|-
| colspan="1" rowspan="1" | Tris-HCl, pH 7.5
| colspan="1" rowspan="1" | 1 M
| colspan="1" rowspan="1" | 2 mL
| colspan="1" rowspan="1" | 500 mM
|-
| colspan="1" rowspan="1" | MgCl2
| colspan="1" rowspan="1" | 1 M
| colspan="1" rowspan="1" | 200 µL
| colspan="1" rowspan="1" | 50 mM
|-
| colspan="1" rowspan="1" | dATP
| colspan="1" rowspan="1" | 100 mM
| colspan="1" rowspan="1" | 40 µL
| colspan="1" rowspan="1" | 1 mM
|-
| colspan="1" rowspan="1" | dCTP
| colspan="1" rowspan="1" | 100 mM
| colspan="1" rowspan="1" | 40 µL
| colspan="1" rowspan="1" | 1 mM
|-
| colspan="1" rowspan="1" | dGTP
| colspan="1" rowspan="1" | 100 mM
| colspan="1" rowspan="1" | 40 µL
| colspan="1" rowspan="1" | 1 mM
|-
| colspan="1" rowspan="1" | dTTP
| colspan="1" rowspan="1" | 100 mM
| colspan="1" rowspan="1" | 40 µL
| colspan="1" rowspan="1" | 1 mM
|-
| colspan="1" rowspan="1" | DTT
| colspan="1" rowspan="1" | 1 M
| colspan="1" rowspan="1" | 200 µL
| colspan="1" rowspan="1" | 50 mM
|-
| colspan="1" rowspan="1" | PEG-8000
| colspan="1" rowspan="1" | -
| colspan="1" rowspan="1" | 1 g
| colspan="1" rowspan="1" | 25&nbsp;%
|-
| colspan="1" rowspan="1" | NAD+
| colspan="1" rowspan="1" | 100 mM
| colspan="1" rowspan="1" | 200 µL
| colspan="1" rowspan="1" | 5 mM
|-
| colspan="1" rowspan="1" | H2O
| colspan="1" rowspan="1" | -
| colspan="1" rowspan="1" | to final volume of 4 mL
| colspan="1" rowspan="1" | -
|}

Mix dNTPs, NAD+, Tris-HCl, MgCl2 and DTT.

Slowly add PEG-8000 to mixture and mix well, until completely dissolved. Add H2O to a final volume of 4 mL.

Prepare aliquots of the 5x isothermal buffer as required, e.g. 100 µL. Store at -20 ˚C. Preparation of 1,33x Assembly Mastermix

Recipe for 25 x 15 µL aliquots:



{|
|-
| colspan="1" rowspan="1" | '''&nbsp;Component'''
| colspan="1" rowspan="1" | '''&nbsp;Concentration'''
| colspan="1" rowspan="1" | '''&nbsp;Amount'''
| colspan="1" rowspan="1" | '''&nbsp;Final concentration (after adding DNA)'''
|-
| colspan="1" rowspan="1" | &nbsp;5x isothermal rxn buffer
| colspan="1" rowspan="1" | &nbsp;5x
| colspan="1" rowspan="1" | &nbsp;100 µL
| colspan="1" rowspan="1" | &nbsp;1x
|-
| colspan="1" rowspan="1" | &nbsp;Taq DNA Ligase
| colspan="1" rowspan="1" | &nbsp;40 U/µL
| colspan="1" rowspan="1" | &nbsp;50 µL
| colspan="1" rowspan="1" | &nbsp;4 U/µL&nbsp;
|-
| colspan="1" rowspan="1" | &nbsp;T5 Exonuclease
| colspan="1" rowspan="1" | &nbsp;1 U/µL
| colspan="1" rowspan="1" | &nbsp;2 µL
| colspan="1" rowspan="1" | &nbsp;4 U/mL
|-
| colspan="1" rowspan="1" | &nbsp;Q5 Hi-Fi DNA Polymerase
| colspan="1" rowspan="1" | &nbsp;2 U/µL
| colspan="1" rowspan="1" | &nbsp;6.25 µL
| colspan="1" rowspan="1" | &nbsp;25 U/mL
|-
| colspan="1" rowspan="1" | &nbsp;H<sub>2</sub>O
| colspan="1" rowspan="1" | 
| colspan="1" rowspan="1" | &nbsp;216.75
| colspan="1" rowspan="1" | 
|-
| colspan="1" rowspan="1" | &nbsp;'''Total volume'''
| colspan="1" rowspan="1" | 
| colspan="1" rowspan="1" | '''375 µL'''
| colspan="1" rowspan="1" | 
|}

Preparation of 1.33x Assembly Mastermix

Work on ice. Mix H2O and 5x buffer, then add enzymes.

Prepare 25 x 15 µL aliquots in PCR tubes. Store at -20 ˚C.

These aliquots are concentrated 1.33 x - add your DNA in a volume of 5 µL to a final volume/concentration of 20 µL / 1x. Gibson assembly

After addition of DNA, incubate Gibson assembly mix at 50 ˚C for 45 min.
[[Category:Protocols]]