Chemically competent E. coli cells


  • Streak E.coli DH10B on solid LB medium and incubate overnight at 37℃


  • Pick up a single colony from the solid culture and start a 10mL liquid LB culture and grow overnight at 37℃
  • Prepare the stock solutions for Day 3 → 5M CH3COOK, 1M MnCl2*4H2O, 1M CaCl2*2H2O, 1M MOPS


  • Add 2.5mL from the liquid culture in a 2L flask with 250mL liquid LB and incubate at 37℃ until OD600=0.5-0.6 (~3h)

    TFBI (pH=5.8) TFBII (pH=6.5)
    CH3COOK 30mM MOPS 10mM
    RbCl 100mM CaCl2*2H2O 75mM
    MnCl2 10mM RbCl 10mM
    CaCl2*2H2O 10mM Glycerol 15%
    Glycerol 15%  

    For every 50mL culture we need 15mL TFBI and 2mL TFBII, so in our case we need 75mL TFBI and 10mL TFBII.

  • When the culture is ready split it in 50mL Falcon tubes and pellet cells at 2500rpm for 10min (4℃)
  • Discard the supernatant and resuspend in 15mL TFBI in each tube
  • Rest on ice for 30min
  • Pellet cells, discard the supernatant and resuspend in 2mL TFBII
  • Aliquot 100μL cells in 1.5ml Eppendorf tubes and freeze it immediately in liquid nitrogen
  • Store aliquots at -80℃

Competent Agrobacterium Tumefaciens cells

The creation of competent Agrobacterium Tumefaciens cells is a 5-day procedure. In our case the Agrobacterium Tumefaciens strain we use is C58C1.

Day 1

  • Streak the preferred Agrobacterium Tumefaciens strain on solid LB medium with rifamycin (50μg/mL)
  • Incubate for 2 days at 28℃ in dark

Day 3

  • Pick up a single colony and start a 5mL liquid culture in LB with rifamycin (50μg/mL)
  • Incubate (shaking) at 28℃ until saturation (overnight)

Day 4

  • Add 100μL of the saturated culture in a 2L flask with 200mL of LB with rifamycin (50μg/mL)
  • Incubate (shaking) overnight at 28℃

Day 5

  • Measure OD600 (should be ~0.8-1)
  • Split culture in 50mL Falcon tubes and pellet cells at 3000rpm for 10 min (4℃)
  • Discard the supernatant
  • Dilute cells in every Falcon in 1mL cold CaCl2 20mM
  • Aliquot 100μL of cells in 1.5mL Eppendorf tubes and freeze them immediately in liquid nitrogen
  • Store at -80℃

E. coli Transformation

We use E.coli DH10B cells for our project’s needs. This protocol’s steps are the following:

  • Rest E.coli DH10Bcells on ice for 3-5 mins
  • Add ~100ng of DNA
  • Rest on ice for 30 min
  • Heat shock at 42℃ for 1 min and thaw on ice for 1 min
  • Add up to 1mL liquid LB
  • Incubate at 37℃ (shaking) for 1h
  • Pellet cells at 2100 rcf for 2 min and discard the supernatant
  • Resuspend the pellet in LB leftovers
  • Plate in selective solid medium and incubate overnight at 37℃

Agrobacterium tumefaciens Transformation

For the purpose of our project we use Agrobacterium Tumefaciens C58C1 strain. The transformation procedure we use is the following:

  • Thaw competent Agrobacterium Tumefaciens C58C1 cells on ice for 30 minutes.
  • Add ~5μg of plasmid DNA and cold shock the cells (freeze in liquid nitrogen)
  • Incubate at 37℃ for 1 hour
  • Add sterile liquid LB up to 1mL and incubate (shaking) at 28℃ for 2-3 hours
  • Pellet the cells (3000g for 2min) and discard the supernatant
  • Resuspend the pellet in LB leftovers and plate in solid selective medium
  • Incubate at 28℃ for 2 days in the dark

Isolation of plasmid DNA (mini-preps)

The process of plasmid isolation can be separated into three main steps: Alkaline Lysis, Ethanol Precipitation and Dilution. For this procedure 3 solutions will be needed: Solution I,II and III.

Solution I Solution II Solution III
Glucose 50mM NaOH 0.2N Potassium Acetate 60mL
Tris-Cl 25mM SDS 1% Glacial Acetic Acid 11.5mL
EDTA 10mM   dH2O 28.5mL

Alkaline Lysis:

  • Pellet cells and discard all of the supernatant (2x)
  • Resuspend the pellet at 100μL of Solution I
  • Add 200μL of Solution II, mix by turnaround and let sit for a maximum of 5min
  • Add 150μL of Solution III and mix by turnaround
  • Centrifuge at 13800g for 10min and collect the supernatant in a clean tube

Ethanol Percipitation:

  • Add 900μL of 100% Ethanol and rest in ice for 2min
  • Centrifuge at 13800g for 20min and discard the supernatant
  • Wash pellet with 60μL 70% Ethanol
  • Centrifuge at 13800g for 5min, discard the supernatant and remove Ethanol completely
  • Air dry in hood for 10min


  • Dilute pellet in 30μL dH2O (sterile)


Agroinfiltration is a procedure in which we inject Agrobacterium Tumefaciens C58C1 containing our prefered gene into plant leaves to make them express this gene’s protein.

The procedure takes 2 days.

Day 1:

  • Start an overnight liquid culture of Agrobacterium Tumefaciens C58C1 (28℃ )

Day 2:

  • Pellet the cells at 2800 rpm for 10 minutes (4℃ )
  • Discard the supernatant and resuspend the pellet in 10mM MgCl2 solution (~5mL)
  • Pellet the cells again, discard the supernatant and resuspend the pellet in 1mL MM solution (10mM MgCl2 and 10mM MES)
  • Measure the concentration of the cells using a photometer and dilute until the absorption at 600nm is 0.5
  • Make a small nick using a small needle at the leaf’s underside
  • Inject a small amount of the cell solution using a syringe

Note: The plant used in this procedure must be unwatered.

Golden Gate Cloning

We used Golden Gate cloning to connect the parts we needed to be expressed together.

This protocol was adjusted as NEB instructed.

For 20μL reaction:

Components Amount
10x T4 buffer 2μL
100x BSA 0.2μL
Backbone Vector 100ng
BsaI (NEB) 1μL
T4 ligase 1μL
Assembly pieces equimolar
dH2O up to 20μL

Thermocycler settings are the following:

Stage Temperature Time
Digestion 37℃ 3 min
Ligation 16℃ 4 min
Final Digestion 37℃ 10 min
Enzyme inactivation 80℃ 5 min

Digestion and Ligation for 25 cycles


For a 50μL reaction

Component Amount
10x Taq Buffer 5μL
dATP 0.5mM 2.5μL
TaqPol 5U/μL 0.2μL
PCR product 25μL
dH2O up to 50μL

Incubate at 72℃ for 30 min

Ligation of A-tailed products

For a 20μL reaction

Components Amount
10x T4 DNA ligase Buffer 2μL
Vector A 1:3 vector to insert ratio
T4 DNA Ligase  1μL
dH2O up to 20μL 

Incubate at room temperature for 2 hours

Colony PCR

For a 50μL reaction

Components Amount
10x Taq Buffer 33.5μL
dNTPs 10mM 5μL
FW Primer 10μM 1μL
REV Primer 10μM 2.5μL
TaqPol 5U/μL 2.5μL
Template 0.5μL
dH2O up to 50μL

Thermocycler settings

Stage Time Temperature
Initial Denaturation 3 min 94℃
Denaturation 45 sec 94℃
Annealing 30 sec Variable
Extension Variable 72℃
Final Extension 5 min 72℃

High Fidelity PCR

We use High Fidelity PCR for a plethora of our experiments and it’s the main method to multiply our parts and constructs.

For a 50μL reaction:

Components Amount
Template DNA 1ng 0.5μL
5x Phusion Buffer 10μL
FW Primer (0.5μM) 2.5μL
REV Primer (0.5μM) 2.5μL
dNTPs (0.2mM) 1μL
Phusion Dna Polymerase (2U/μL) 0.5μL
dH2O up to 50μL

Restriction Digestions

In our case we used the restriction enzymes NdeI and SacI. We used the protocol provided by NEB. For a 50μL reaction:

Component Amount
DNA 1μg
10x Buffer 5μL
NdeI 1μL
SacI 1μL
dH2O up to 50μL

Incubate for 5-15 min at 37℃