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− | 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 | + | For screening a large number of clones, single colonies were dissolved in 20 µl LB medium of which 1 µl was used as template for the PCR. |
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<li>15 µl aliquot of master mix | <li>15 µl aliquot of master mix | ||
<li>0.02-0.5 pmol DNA in total for 2-3 fragments<br> | <li>0.02-0.5 pmol DNA in total for 2-3 fragments<br> | ||
− | + | <ul> or </ul> | |
<li>0.2-1 pmol DNA in total for 4-6 fragments | <li>0.2-1 pmol DNA in total for 4-6 fragments | ||
<li>Fill up with water to 20 µl | <li>Fill up with water to 20 µl |
Revision as of 17:54, 16 October 2016
Experimental: Protocols, Methods and Material
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:
Temperature |
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:
Temperature |
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 as template for the PCR.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:
Temperature |
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 |
Thermocycling Conditions:
Temperature |
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:
Temperature |
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 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
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
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
- 2-3 times more insert than backbone (molar ratio)
- 5 times more insert for fragments < 200 bp (molar ratio)
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.
-