Difference between revisions of "Team:ETH Zurich/Experiments"

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Restreak all experiments you want to run from cryostocks
 
Restreak all experiments you want to run from cryostocks
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</li><h4>Day 0: Prepare precultures</h4>
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Prepare precultures of all the experiments you want to run. Prepare 5mL of freshly prepared M9 medium (prepared the same morning), necessary antibiotics, and pick one colony from the plate. Ideally you want to run three biological replicates, but this can be done in three different experiments (the 96-well plate has a finite number of wells and your experiment might not fit). Priority is technical replicates (see setting up the experiment). Keep M9 the in +4 frigde until next day.
 
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Revision as of 18:18, 19 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 resuspended 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 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. In order to obtain high base exchange efficiency, the template need to be removed from the reaction thoroughly.

Isothermal "Gibson" Assembly2

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.
  • Centrifuge for 5 min (4000xg, 4°C)
  • Carfully discard supernatant, keep cells always on ice.
  • Resuspend the cells in 4 ml ice-cold TFB2 buffer.
  • Prepare aliquots of 100 µl in pre-cooled, sterile microcentrifuge tubes and freeze in liquid nitrogen. Store the competent cells at -80° C

Transformation Protocol for Chemically Competent Cells

  • Thaw the competent cells on ice
  • Use 50 ul of the competent cells for one transformation, do not add more than 5 ul of DNA to the cells (10% of the volume of the competent cells)
  • Incubate them for 20 min on ice
  • Incubate them for 90 s at 42° C and add afterwards 500 µl of SOC
  • Let them regenerate for 1 h at 37° C with shaking
  • Plate a part of the culture according to your expectations on LB-agar plates with the approbriate antibiotic.

Preparation of Electrocompetent Cells

  • Inoculate 600 ml of prewarmed LB medium with 6 ml overnight culture and grow the bacteria to an OD600 of 0.4.
  • Cool the culture on ice fpr 30 min, transfer 300 ml of the culture into 50 ml tubes and centrifuge them for 10 min (3000xg, 4°C). Discard supernatant and add the rest of the culture. Again discard supernatant.
  • Carfully resuspend cells in ice-cold, autoclaved and deionized water and fill up to 50 ml
  • Centrifuge again for 10 min (3000xg, 4°C) and repeat wash step from above
  • Carfully resuspend cells in ice-cold 10% glycerol (working always on ice)
  • Centrifuge for 10 min (3000xg, 4°C)
  • Discard supernatant
  • Resuspend the cells in the remaining glycerol solution.
  • Prepare aliquots of 35 µl in pre-cooled, sterile microcentrifuge tubes and freeze in liquid nitrogen. Store the competent cells at -80° C

Transformation Protocol for Electrocompetent Cells

  • Thaw the electrocompetent cells on ice. Pre-chill also the electroporation cuvette.
  • Add DNA to the cells. Attention: the DNA should contain as little ions as possible. Purify or dilute reactions containing salt.
  • Transfer the bacteria/DNA mix into a pre-chilled electroporation cuvette and electroporate them.
  • Add IMMEDIATELY 500 µl of SOC and transfer them back into the microcentrifuge tube.
  • Let them regenerate for 1 h at 37° C with sufficient shaking.
  • Plate a part of the transformation mix according to your expectations on LB-agar plates with the approbriate antibiotic.

Buffer and Medium

TFB1

Component Concentration Amount per 100 ml
RbCl 100 mM 1.21 g
MnCl2 · 4 H2O 50 mM 0.99 g
Potassium acetate · 4 H2O 30 mM 0.29 g
CaCl2 · 2 H2O 10 mM 0.147 g
Glycerol 15 % 11.9 g


Adjust pH to 5.5 and sterilize by filtration

TFB2

Component Concentration Amount per 100 ml
MOPS 100 mM 0.0047 g
RbCl 50 mM 0.0027 g
CaCl2 · 2 H2O 75 mM 0.024 g
Glycerol 15 % 0.27g


Adjust pH to 6.5 and sterilize by filtration

LB Medium

Component Concentration Amount per 1l
Bacto-tryptone 1% 10g
Yeast Extract 0.5% 5g
NaCl 1% 10g


Sterilized by autoclaving

LB Agar

Component Concentration Amount per 1l
Bacto-tryptone 1% 10g
Yeast Extract 0.5% 5g
NaCl 1% 10g
Agar-agar 1.5% 15g


Sterilized by autoclaving

M9 Salts 10x

Component Concentration Amount per 250ml
Na2PO4· 7 H2O 128g/L 31.96g
KH2PO4 30 g/L 7.64g
NaCl 5g/L 1.248g
NH4 10g/L 2.495g


Sterilized by autoclaving

LIV solution

Component Concentration Amount per 10ml
L-Leu 10g/L 0.1g
L-Ile 3 g/L 0.03g
L-Val 15g/L 0.15g


Sterilized by filtration

Trace elements solution US*

Component Concentration Amount per 50ml
FeSO4 5.5g/L 0.25
CaCl2· 2 H2O 4.1g/L 0.2g
MnCl2· 4 H2O 1.5g/L 0.075g
ZnSO4 1.05g/L 0.0502g
H3BO3 0.3g/L 0.015g
Na2[MoSO4]· 2 H2O 0.25g/L 0.013g
CuCl2· 2 H2O 0.15g/L 0.0075g
Na2[EDTA]· 2 H2O 0.84g/L 0.042g


Sterilized by filtration

M9* LIV Medium

Component Concentration Volume for 1L
M9 Salts 10x 100ml
Glucose 50% 20ml
MgSO4 1M 2ml
CaCl2 0.1M 1ml
Antibiotic 1000x 1ml
Trace elements solution US* 1000x 1ml
Thiamine 10g/L 2ml
LIV Solution 100x 10ml
H2O 55.5M 863ml

HEPES Buffer

Component Concentration Amount per 50ml
NaOH 5M 10g
HEPES 100mM 0.475g


Adjust pH to 7.0 and sterilize by filtration

Isothermal Reaction Buffer

Component Concentration Amount per 15ml
Tris-HCl pH7.5 1M 3ml
MgCl2 2M 150ul
dNTP 100mM 60ul
DTT 1M 300ul
NAD 100mM 300ul
PEG N/A 1.5g


Sterilized by filtration

Gibson Assembly Mix

Component Concentration Amount per 15ml
ITA Buffer 3.5x 350ul
H2O 55.5M 700ul
T5 Exonuclease 10,000units/ml 0.64ul
Phusion DNA Pol 2,000units/ml 20ul
Taq DNA Ligase 40,000units/ml 160ul


Transferred into PCR tubes in 15ul aliquots

DETA/NO 650mM Solution

Component Concentration Amount per 1ml
DETA/NO N/A 10mg
NaOH 0.1M Up to 1ml

AHL Solution 100mM

Component Concentration Amount per 1ml
N-Hexanoyl-L-Homoserine Lactone N/A 19.9mg
DMSO N/A Up to 1ml

Plate reader experiment

  • Day -1: Restreak

    Restreak all experiments you want to run from cryostocks

  • Day 0: Prepare precultures

    Prepare precultures of all the experiments you want to run. Prepare 5mL of freshly prepared M9 medium (prepared the same morning), necessary antibiotics, and pick one colony from the plate. Ideally you want to run three biological replicates, but this can be done in three different experiments (the 96-well plate has a finite number of wells and your experiment might not fit). Priority is technical replicates (see setting up the experiment). Keep M9 the in +4 frigde until next day.

References:

  • [1] Tom Richard, Department of Agricultural and Biological Engineering, Penn State University.
  • [2] Gibson, Daniel G., et al. "Enzymatic assembly of DNA molecules up to several hundred kilobases." Nature methods 6.5 (2009): 343-345.

Thanks to the sponsors that supported our project: