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 competent E. coli

1. Thaw the competent cells on ice
2. Add 1 μL DNA (0.2 - 200 ng) to 50-100 μL competent cells
3. Leave sample on ice for approximately 20 min
4. Heat shock the cells for 90 s at 42 °C
5. Add 500 μL of SOC to the sample
6. Let the cells recover for 60 min at 37 °C, 220 rpm
7. Plate appropriate amount of cell suspension (50 - 200 μL) on LB-agar-plates containing the appropriate antibiotic
8. Let bacteria grow overnight at 37 °C

According to qiagen DNA protocols & application

Plasmid preparation

Day before: Preparation of a preculture in LB-medium containing the appropriate antibiotics. The amount of cell culture required for plasmid preparation depends on the copy number of the plasmid (between 5 and 20 mL)

1. Centrifuge the preculture in appropriate tubes for 10 min at 4000 g
2. Carefully remove the supernatant
3. Resuspend pellet in 200 μL resuspension solution
4. Add 200 μL lysis solution and invert the tube gently 1 to 2 times
5. Lysis should not exceed 5 min
6. Add 350 μL neutralization solution and invert the tube 4 to 6 times
7. Centrifuge the suspension for 10 min at 12 000 rcf
8. Prepare the columns by adding 500 μL of column preparation solution and centrifuging it for 1 min at 12 000 rcf. Discard the flow-through
9. Transfer the supernatant of the centrifuged samples onto columns
10. Spin for 1 min at 12 000 rcf and subsequently discard the flow-through
11. Add 750 μL wash solution and spin the loaded column for 1 min at 12 000 rcf. Discard the flow-through
12. Dry the columns by centrifuging them for 1 min at 12 000 rcf
13. Place columns in new collection tubes
14. Elute the plasmid DNA with 50 μL ddH₂O to increase the plasmid concentration

The Sigma-Aldrich Plasmid Miniprep Kit was used

Preparation of samples for sequencing at Microsynth

Add 12 μL DNA (60-100 ng/μL) to 3 μL of the corresponding primer (10 μM).

Preparation of antibiotics stock

Preparation of 1000x stock solutions

• Ampicillin (200 mg/mL) in H₂O, sterile filtered
• Kanamycin (50 mg/mL) in H₂O, sterile filtered
• Chloramphenicol (34 mg/mL) in ethanol
• Tetracycline (10 mg/mL) in ethanol
• Streptamycin?

PCR procol for phusion DNA polymerase

Restriction Endonuclease Reactions

Enzymes, buffers and protocol are from New England BioLabs

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 min.

Enzymes, buffers and protocol are from New England BioLabs

DNA Purification by Centrifugation

A. Dissolving the Gel Slice

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

or

B. Processing PCR Amplifications

Add an equal volume of Membrane Binding Solution to the PCR mix.

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 min.
3. Centrifuge at 16,000 rcf for 1 min. Discard flowthrough and reinsert Minicolumn into Collection Tube.
4. Add 700 µl Membrane Wash Solution (ethanol added). Centrifuge at 16,000 rcf for 1 min. Discard flowthrough and reinsert Minicolumn into Collection Tube.
5. Repeat the step before with 500 µl Membrane Wash Solution. Centrifuge at 16,000 rcf for 5 min.
6. Empty the Collection Tube and recentrifuge the column assembly for 1 min with the microcentrifuge lid open (or off) to allow evaporation of any residual ethanol.
7. Carefully transfer Minicolumn to a clean 1.5 ml microcentrifuge tube.
8. Add 50 µl of Nuclease-Free Water to the Minicolumn. Incubate at room temperature for 1 min. Centrifuge at 16,000 rcf for 1 min.
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

Preparation of a 5 cm x 5 cm gel

1. Add 0.25 g Agarose to 25 mL TAE (0.5x) and heat up in microwave to dissolve it
2. Let the solution cool down to approximately 50 °C
3. Add nucleic acid dye (e.g. 2.5 μL peqGREEN) and mix
4. Pour the solution in a tray using an appropriate comb
5. Add loading dye (e.g. NEB purple loading dye) to the samples
6. Fill the samples and ladder in the wells
7. Run the gel at 135 V

Preparation of cryostocks

1. Cultivate bacteria (37 °C) in medium with antibiotic (e.g. 5 mL 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

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.

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

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

Reaction Mixture Recipe: 6 ml 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