Polymerase Chain Reaction - for Construction

New England Biolabs Phusion High-Fidelity DNA Polymerase

Composition:

Component	50 µl Reaction
Phusion HF Buffer (5x)	10 µl
dNTPs (10 mM)	1 µl
Forward Primer (10 µM)	2.5 µl
Reverse Primer (10 µM)	2.5 µl
Template DNA	variable
DMSO (optional)	up to 3%
Nuclease-free water	up to 50 µl
Phusion Polymerase	0.5 µl

Thermocycling Conditions:

Temperatur	Time
98°C	30 seconds
98°C 45-72°C 72°C	5-10 seconds 10-30 seconds 15-30 seconds per kb
72°C	5-10 minutes

The appropriate annealing temperature was calculated from NEB's Tm Calculator

Kapa Biosystems Hifi Hotstart Ready Mix

Component	50 µl Reaction
Master Mix (2x)	25 µl
Forward Primer (10 µM)	2.5 µl
Reverse Primer (10 µM)	2.5 µl
Template DNA	variable
Nuclease-free water	up to 50 µl

Thermocycling Conditions:

Temperatur	Time
95°C	3 minutes
98°C 55-75°C 72°C	20 seconds 15 seconds 15-60 seconds per kb
72°C	1 minute per kb

Kapa Hifi Hotstart has similar annealing temperatures as Phusion DNA polymerase, even slightly higher. Only in very few cases a lower annealing temperature was found to be better.

Polymerase Chain Reaction - Colony PCR

For screening a large number of clones, single colonies were dissolved in 20 µl LB medium of which 1 µl was used for the PCR as template.

Solis BioDyne FirePol DNA Polymerase

Composition:

Component	8 x 20 µl Reaction (+ 10 µl excess)
FIREPol DNA Polymerase	0.85 µl
MgCl2 (25 mM)	10.2 µl
Reaction Buffer B (10x)	17 µl
dNTPs (10 mM)	3.4 µl
Forward Primer (10 µM)	3.4 µl
Reverse Primer (10 µM)	3.4 µl
Template DNA	1 µl per 20 µl reaction
Nuclease-free water	123.25 µl

Thermocycling Conditions:

Temperatur	Time
98°C	3-5 minutes
95°C 50-72°C 72°C	30-60 seconds 30-60 seconds 1 minute per kb
72°C	5-10 minutes

New England Biolabs Taq DNA Polymerase

Composition:

Component	8 x 20 µl Reaction (+ 10 µl excess)
Taq Reaction Buffer (10x)	17 µl
dNTPs (10 mM)	3.4 µl
Forward Primer (10 µM)	3.4 µl
Reverse Primer (10 µM)	3.4 µl
Template DNA	1 µl per 20 µl reaction
Taq DNA Polymerase	0.85 µl
Nuclease-free water	133.45 µl

Create a mastermix, aliquote into PCR tubes and add 1 µl the template (resuspended colony from plate in 20 µl LB medium).

Thermocycling Conditions:

Temperatur	Time
95°C	5 minutes
95°C 45-68°C 68°C	30 seconds 20 seconds 1 minute per kb
72°C	5 minutes

The appropriate annealing temperature was calculated from NEB's Tm Calculator

Site-Directed Mutagenesis (QuickChange)

Component	50 µl Reaction
Kapa Hifi Hotstart Master Mix (2x)	25 µl
Forward Primer (10 µM)	2.5 µl
Reverse Primer (10 µM)	2.5 µl
Template DNA	variable
Nuclease-free water	up to 50 µl

Thermocycling Conditions:

Temperatur	Time
95°C	3 minutes
98°C 65°C 72°C	20 seconds 15 seconds 15-60 seconds per kb
72°C	1 minute per kb

Primers were designed according to the guidlines of The Richard Lab ¹

• The targeted mutation should be included into both primers.
• The mutation can be as close as 4 bases from the 5-terminus.
• The mutation should be at least 8 bases from the 3-terminus.
• At least eight non-overlapping bases should be introduced at the 3-end of each primer.
• At least one G or C should be at the end of each primer.
• Design your primers (including the mutations) to have a Tm >=78°C.

The resulting PCR product has to be purified (e.x. Agencourt AMPure XP) and digested with DpnI (NEB) for 4 hours and gel-purified. In case Phusion polymerase is used, DpnI can directly be added to the PCR. The product can then immediately be used for transformation. With proper removal of template plasmid DNA the efficiency of exchanged bases is very high.

Isothermal "Gibson" Assembly

Recipe for Ready-to-Use Isothermal Assembly Mixes

5x Isothermal Reaction Buffer (6ml)

• 3 ml of 1 M Tris-HCl pH 7.5
• 150 µl of 2 M MgCl2
• 60 µl of 100 mM dGTP
• 60 µl of 100 mM dATP
• 60 µl of 100 mM dTTP
• 60 µl of 100 mM dCTP
• 300 µl of 1 M DTT
• 1.5 g PEG-8000
• 300 µl of 100 mM NAD

This buffer can be aliquoted and stored at -20 °C.

Isothermal Assembly Master Mix

• 320 µl 5x isothermal reaction buffer
• 0.64 µl of 10 U/µl T5 exonuclease
• 20 µl of 2 U/µl Phusion DNA polymerase
• 160 µl of 40 U/µl Taq DNA ligase
• Fill up with water to a final volume of 1.2 ml

The master mix is devided into aliquots of 15 µl and stored at -20 °C.

Protocol for Isothermal Assembly

• 15 µl aliquot of master mix
• 0.02-0.5 pmol DNA in total for 2-3 fragments
  or
• 0.2-1 pmol DNA in total for 4-6 fragments
• Fill up with water to 20 µl

Consider the following:

• 2-3 times more insert than backbone (molar ratio)
• 5 times more insert for fragments < 200 bp (molar ratio)

The assembled mix is then incubated for 60 minutes at 50 °C. It can be directly transformed with chemically competent cell (volume of assembly reaction not exceeding 10% of the volume of the competent cells). Otherwise, the reaction mix is purified and desalted (e.x. Agencourt AMPure XP) and a fraction of it transformed with electrocompetent cells.

Preparation of Competent Cells

Preparation of Chemically Competent Cells

• Inoculate 100 ml of prewarmed LB medium with 1 ml overnight culture and grow the bacteria to an OD₆₀₀ of 0.5.
• Cool the culture on ice, transfer the cells into cetrifugation tubes and harvest them by centrifugation for 5 min (4000xg, 4°C)
• Carfully discard supernatant, keep cells always on ice.
• Resuspend cells in 30 ml cold TFB1 and incubate on ice for 90 minutes.

• Thanks to the sponsors that supported our project:

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