Team:ETH Zurich/lab/protocols

Preparation of competent E. coli

Day before: Preparation of a Top10 cells preculture in LB-medium containing streptomycin (25 mg/L)

1. Addition of 1 mL overnight preculture to 100 mL LB-medium + streptomycin (25 mg/L)

2. Cultivate culture at 37 °C, 220 rpm until it reaches an OD600 of 0.5

3. Cool culture for 5 min on ice and centrifuge it for 5 min at 4 °C, 4000 g

4. Discard the supernatant and resuspend the cells in cold TFB1 buffer (30 mL, 4 °C)

5. Keep the suspension on ice for 90 min

6. Centrifuge the suspension for 5 min at 4 °C, 4000 g and discard the supernatant

7. Resuspend the cells in 4 mL cold TFB2 buffer

8. Make aliquots of 100 μL and freeze the aliquots in dry ice in ethanol

9. Store aliquots at -80 °C

According to qiagen DNA protocols & application

Preparation of DNA from iGEM kit

Addition of 10 μL H₂O to appropriate well, wait for 5 min, transfer into sterile tube

Transformation of E. coli

Addition of 1 μL DNA (0.2 - 200 ng) to 75 μL competent cells (thawed on ice), put samples on ice for approximately 20 min Heat shock: 90 s at 42 °C Addition of 500μL SOC to samples, let cells recover for 60 min at 37 °C, 220 rpm Plate on LB-agar-plates containing the appropriate antibiotic (ampicillin (200 mg/L) or chloramphenicol (34 mg/L)) Let bacteria grow overnight at 37 °C According to qiagen DNA protocols & application

Precultures for minipreps

Pick colonies of transformed bacteria, grow overnight at 37 °C, 220 rpm in 5 mL LB-medium with corresponding antibiotic

Miniprep

The Sigma-Aldrich Plasmid Miniprep Kit was used. DNA was eluted with 50 μL ddH₂O to increase the plasmid concentration.

Preparation of samples for sequencing at microsynth

12 μL DNA (conc 60-100ng/μL) if DNA conc too high, dilute appropriately with H2O 3 μL corresponding primer ==Preparation of antibiotics stock:

2 g Ampicillin were dissolved in 10 mL H2O and sterile filtered 0.5 g Kanamycin were dissolved in 10 mL H2O and sterile filtered 0.34 g Chloramphenicol were dissolved in 10 mL Ethanol

PCR procol for phusion DNA polymerase

Components 20μL reaction 50 μL reaction Nuclease-free water to 20μL to 50μL 5x Phusion HF buffer 4 μL 10 μL 10mMdNTPs 2μL 5μL 10μM Forward Primer 1μL 2.5μL 10μM Reverse Primer 1μL 2.5 μL Template DNA 1μL 1μL DMSO 0.6 μL 1.5μL Phusion DNA polymerase 0.2 μL 0.5 μL

Optimizing Restriction Endonuclease Reactions

1μL Restriction enzyme 1μL Restriction enzyme 0.5μL Restriction enzyme 0.5μL Restriction enzyme 20 μL template DNA 5μL Cut Smart Buffer fill up with 22 μL H2O to 50μL total volume

Dephosphorylation of 5’-ends of DNA using CIP

Add 1 unit of CIP for every 1 pmol of DNA ends (about 1 μg of a 3 kb plasmid) and incubate at 37°C for 30–60 minutes. Purify DNA by spin-column centrifugation, proceed with ligation. According to New England BioLabs protocol

DNA Purification by Centrifugation

A. Dissolving the Gel Slice 1. Following electrophoresis, excise DNA band from gel and place gel slice in a 1.5ml microcentrifuge tube. 2. Add 10µl Membrane Binding Solution per 10mg of gel slice. Vortex and incubate at 50–65°C until gel slice is completely dissolved.

or

B. Processing PCR Amplifications 1. Add an equal volume of Membrane Binding Solution to the PCR amplification.

Binding of DNA 1. Insert SV Minicolumn into Collection Tube. 2. Transfer dissolved gel mixture or prepared PCR product to the Minicolumn assembly. Incubate at room temperature for 1 minute. 3. Centrifuge at 16,000 × g for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube. Washing 4. Add 700µl Membrane Wash Solution (ethanol added). Centrifuge at 16,000 × g for 1 minute. Discard flowthrough and reinsert Minicolumn into Collection Tube. 5. Repeat Step 4 with 500µl Membrane Wash Solution. Centrifuge at 16,000 × g for 5 minutes. 6. Empty the Collection Tube and recentrifuge the column assembly for 1 minute with the microcentrifuge lid open (or off) to allow evaporation of any residual ethanol. Elution 7. Carefully transfer Minicolumn to a clean 1.5ml microcentrifuge tube. 8. Add 50µl of Nuclease-Free Water to the Minicolumn. Incubate at room temperature for 1 minute. Centrifuge at 16,000 × g for 1 minute. 9. Discard Minicolumn and store DNA at 4°C or –20°C.

According to Promega Wizard SV Gel and PCR Clean-Up System, Quick protocol

Agarose gel electrophoresis

0.5 g Agarose in 50 mL TAE (0.5x), heat in microwave to dissolve let solution cool down to approximately 50 °C, add 4 μL peqGREEN, mix pour solution in tray, use appropriate comb add 10μL NEB purple loading dye (6x) to samples fill samples and ladder (10 μL) in wells run gel at 135 V for 50min ! NEB purple loading dye runs different with different agarose concentrations

Preparation of cryostocks

1. Cultivate bacteria (37 °C) in medium with antibiotic (e.g. 5mL LB, 5μL amp + 500μL preculture) until they are in log phase (OD=0.8-1.2) 2. Add 750 μL sterile glycerol (30%) to 750 μL bacteria culture in a screw top tube 3. Freeze the glycerol stock tube at -80 °C

Quik change mutagenesis

14 μL H2O 2 μL HF buffer 1.6 μL dNTPs 0.5 μL of primer 1 0.5 μL of primer 2 0.4 μL Phusion polymerase 1 μL template DNA (2-20 ng)

18 cycles, te: 2 min/kb

Digestion of the template DNA: Addition of 1 μL DpnI, 1h at 37 °C

Heat inactivation of DpnI: 20 min at 80 °C

For transfomation add 5 μL of reaction mix to 75 μL of competent cells

Gibson Assembly

Reagents: T5 exonuclease - Epicentre Phusion DNA polymerase - Finnzymes Taq DNA ligase - NEB

One-step isothermal DNA assembly protocol.

 1.  PCR up the DNA fragments to be cloned (each primer overhang is min 20 nt).
 2.  Cut open the backbone.
 3.  Heat inactivate the enzymes if cut open an empty vector OR

Gel purify the cut backbone if cut open a full vector.

 4.  Gel purify the PCR fragments.
 5.  Mix the backbone and PCR fragments in max 5µl total volume, in molar ratio 1:1.
 6.  Thaw reaction mixture on ice.
 7.  Add DNA mixture (5µl) to the reaction mixture (15µl). Reaction mixture in -20C freezer in 3.40.
 8.  Heat the PCR machine at 50°C.
 9.  Run the reaction for 1 hour at 50°C.
 10. Transform into bacteria (1-5ul or more if necessary).

Here is an online Gibson Assembly tool. http://django.gibthon.org/

Reaction Mixture Recipe: 6ml of 5x isothermal reaction buffer were prepared by combining:

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.

An assembly master mixture was prepared by combining: 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 water up to a final volume of 1.2 ml

This reagent-enzyme mix is aliquoted (15 µL aliquots) and stored at -20 °C. This mixture can tolerate numerous freeze-thaw cycles and remains stable even after one year. The exonuclease amount is ideal for the assembly of DNA molecules with 20–150 bp overlaps. For DNA molecules overlapping by greater than 150 bp, 3.2 µl of 10 U/µl T5 exonuclease was used to prepare the assembly master mixture above. Frozen 15 µl assembly mixture aliquots were thawed and then kept on ice until ready to be used. Five microliters of the DNA to be assembled were added to the master mixture in equimolar amounts. Between 10 and 100 ng of each 6 kb DNA fragment was added. For larger DNA segments, proportional amounts of DNA were added (for example, 250 ng of each 150 kb DNA segment). Incubations were performed at 50 °C for 15 to 60 min (60 min was optimal).

Preparation of alginate beads

1 bead: approximatly 14.5 µl alginate-NaCl 100 bacteria/bead: Harvest bacteria in exponential phase (OD between 1.5 and 2.0), measure OD Resuspend bacteria in sterile NaCl solution (0.9%) Add 2.5 ml of the bacteria NaCl suspension to 10 ml alginate (2.5%) to reach

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