• Pellet 10 mL of over-night liquid culture grown in YPD broth in a 1.5 mL tube by centrifugation at 14,000 x g for 2 minutes.
• Remove the supernatant.
• Resuspend the cells in 90 μL of 50 mM EDTA.
• Add 10 μL of 1000u lyticase and pipet 4 times to mix.
• Incubate the sample at 37°C for 60 minutes to digest the cell wall.
• Centrifuge the sample at 14,000 × g for 2 minutes and then remove the supernatant.
• Add 300 μl of Nuclei Lysis Solution to the cell pellet and pipet to mix.
• Add 100 μl of Protein Precipitation Solution and vortex at high speed for 20 seconds.
• Let the sample sit on ice for 5 minutes.
• Centrifuge at 14,000 × g for 3 minutes.
• Transfer the supernatant containing the DNA to a clean 1.5 ml tube containing 300 μl of room temperature isopropanol.
• Gently mix by inversion until the DNA is visible.
• Centrifuge at 14,000 × g for 2 minutes.
• Carefully decant the supernatant and drain the tube on clean absorbent paper.
• Add 300 μl of room temperature 70% ethanol and invert the tube several times to wash the DNA pellet.
• Centrifuge at 14,000 × g for 2 minutes.
• Drain the tube on clean absorbent paper and allow the pellet to air-dry for 15 minutes.
• Add 50 μl of DNA Rehydration Solution.
• Add 1.5μl of RNase Solution to the purified DNA sample. Vortex the sample for 1 second and incubate at 37°C for 15 minutes.
• Rehydrate the DNA by incubating at 65°C for 1 hour. Periodically mix the solution by gently tapping the tube.
• Store the DNA at 2–8°C.

Generating electrocompetent cells

This cell preparation describes an innovative and quick methode to generate competent yeast cells

Materials:

• 250 mL YPD medium
• 50 mL ice-cooled BEDS
• 5 mL ice-cooled dithiothreitol (DTT)-Solution
• 150 mL cooled 10 % glycerine
• 10 pre-cooled 50 mL Falcons (-18°C)

Protocol:

• Cultivate a overnight culture of the yeast cells in 50-mL YPD medium at 30°C (120 rpm).
• Ddilute the overnight culture an A600 of 0.15–0.20 in a volume of 50 mL YPD in a flask large enough to provide good aeration

• Incubate 250 mL cells to desired OD₆₀₀ = 0.8-0.9
• Centrifuge cells for 5 min (room temperature, 500g)
• Resuspend in 9 mL ice-cooled (2-4°C))BEDS and 1 mL ice-cooled 1.0 M dithiothreitol (DTT)-Solution
• Incubate for 5 min with gently shaking at 100 rpm at room temperatur
• Centrifuge cells for 5 min (room temperature, 500g)
• Resuspend cells in 1 mL BEDS
• Divide the resuspended cells in 150 μL aliquots (now the cells are ready to use)
• freeze cells slowly at -80°C (Don't freeze in liquid N₂)
• Place in -80°C freezer until needed

Molecular genetical methods

Arabidopsis thaliana: Total RNA Isolation

The frozen plant material has to be grinded in a precooled mortar in liquid nitrogen. About 120 mg of pulverized plant material are transfered into a precooled 2 ml Eppendorf tube and kept frozen until the following steps:

• Add 0.5 ml lysis buffer and immediately homogenize through rough shaking.
• Add 0.5 ml of saturated phenol and mix strongly.
• Add 0.5 ml of chloroform isoamyl alcohol (24:1) and vortex again at high speed for at least 30 seconds.
• Centrifugate for 5 min at 13,000 rpm.
• The lower phase contains now lipids and lipophilic compounds. The upper phase contains nucleic acids (~ 550 µl) and has to be carefully transferred into a new 2 ml Eppendorf tube. This tube has to be filled with 0.5 ml saturated phenol and 0.5 ml chloroform isoamyl alcohol (24:1). Mix immediately.
• Centrifugate at 13,000 rpm for 3 minutes.
• Prepare a new 2 ml Eppendorf tube with 1 ml of chloroform isoamyl alcohol (24:1). Transfer the upper aqueous phase (~ 540 µl) containing the protein purified nucelic acids into the new tube and vortex strongly.
• Centrifugate at 13,000 rpm for 3 minutes.
• Prepare a new 1.5 ml Eppendorf tube with 0.5 ml of pure isopropanol. For the last time transfer the upper phase (~ 400 µl) into the new tube and mix gently.
• Incubate the mixture over night at -20°C. The nucleic acids will precipitate.
• Centrifugate the samples at 13,000 rpm for 15 minutes at 4°C.
• Discard the supernatant and resuspend the pellet in 375 µl sterile H₂O.
• Add 125 µl 8 M lithium chloride and incubate for 2 hours on ice at 4°C. At this point most of the RNA is going to be precipitated.
• Centrifugate at 13,000 rpm at 4°C and discard the supernatant.
• Wash the pellet with 100 µl 70% (v/v) ethanol and discard it after centrifugation.
• Dry the pellet at room temperature.
• Dissolve the pellet in sterile H₂O (~ 25 µl, depending on the size of the pellet).
• Check the quantity and quality of the RNA with a Nanodrop spectrophotometer before starting with a cDNA synthesis.

Arabidopsis thaliana: cDNA Synthesis

After a successful total RNA isolation the RNA has to be translated in cDNA through RT-PCR:

• Take 3 µg/µl of total RNA and add sterile H₂ to 8 µl.

Additionally add

• Vortex and centrifugate shortly.
• Incubate the samples for 10 minutes at 70°C.
• Immediately transfer the samples into ice water for 5 minutes.
• After cooling the samples centrifugate shortly.
• To start the synthesis add

• Mix the samples and centrifugate shortly.
• Incubate for 1 hour at 42°C to translate the RNA into cDNA.
• Transfer the samples to 70°C for 15 minutes to stop the reaction.
• The new synthesized cDNA can be used for PCR after diluting 1:10 with water. Store the cDNA at -20°C.

Ethanol precipitation to clean DNA

To get rid of distracting salts the DNA has to be cleaned. For this we used the following protocol:

• If the volume of the sample containing the DNA is less than 200 µl bring the volume up to 200 µl.
• Add 1/10th volume of 3M sodium acetate and mix.
• Now add 2 volumes of -20°C cold 100% ethanol and vortex for 10 seconds.
• The sample can now be placed in a -20°C freezer overnight or incubated for 30 minutes at -80°C.
• Centrifugate for 10 minutes at 4°C.
• Discard the supernatant containing the ethanol.
• Wash the pellet with 500 µl 4°C cold 70% ethanol by rolling the sample gently.
• Discard the supernatant.
• Let the pellet dry at room temperature or speedvac the pellet.
• Resuspend the Pellet in water (amount is depending on the size of the pellet).

PCR for A.thaliana laccase ampflification

Transformation via electroporation

• Thaw 50 µL competent E.coli cells on ice, dilute with icecold 50 µL glycerine (10 %) if necessary
• Add 0.5-5 µL plasmid to 50 µl electrocompetent cells
• Store cells on ice for 1 minute
• Electroporate at U = 2.5 kV, C = 25 µF, R = 400 Ώ
• Transfer transformation reaction to 450 µL SOC-Medium and shake 1 h at 37 °C
• Centrifuge 2 min at 800 rpm and plate on selective LB-Medium

Transformation of Single Step (KRX) Competent Cells by Promega

Using [http://www.promega.com/~/media/Files/Resources/Protocols/Technical%20Bulletins/101/Single%20Step%20Competent%20Cells%20Protocol.ashx protocol E. coli KRX single step competent cells by Promega]

• Remove Single Step (KRX) Competent Cells from –70 °C, and place on ice for 5 minutes or until just thawed.
• Add 1–50 ng of DNA (in a volume not greater than 5 μL) to the Single Step (KRX) Competent Cells. Move the pipette tip through the cells while dispensing. Quickly flick the tube several times. Do not vortex!
• Immediately return the tubes to ice for 5–30 minutes
• Heat-shock cells for 15–20 seconds in a water bath at exactly 42 °C. Do not shake.
• Immediately place the tubes on ice for 2 minutes.
• Add 450 μL of room-temperature SOC-Medium to each transformation reaction, and incubate for 60 minutes at 37 °C with shaking (approximately 225 rpm). For best transformation efficiency, lay the tubes on their sides and tape them to the platform.
• For each transformation reaction, we recommend plating 100 μL of undiluted cells and 1:10 and 1:100 cell dilutions on antibiotic plates. Incubate the plates at 37 °C over night.

Standard BioBrick Assembly

modified from [http://openwetware.org/wiki/Silver:_BB_Strategy Silver lab]: This assembly method can be used for BioBricks which are bigger than 150 bp. The BioBrick should be at least 500 bp bigger or smaller than the backbone. The BioBrick, which complies with these conditions, is used as the insert and is assembled into the prefix or suffix of the other used BioBrick, called vector. So you have to differentiate between a prefix and a suffix insertion.

Suffix Insertion

• Digestion of insert: at least 700 ng DNA / 10 µL volume, 1 µL 10x Tango buffer, 0.5 µL XbaI, 1 µL PstI. Digest for 2 h to 4 h at 37 °C, afterwards inactivation for 20 min at 80 °C. Clean up the insert via gel electrophoresis. When cutting the insert out of the gel try to avoid staining or exposure to ultraviolet light of the insert.
• Digestion of vector about 700 ng DNA / 10 µL volume, 1 µL 10x orange buffer, 0.5 µL SpeI, 0.5 µL PstI. Digest for 2 h to 4 h at 37 °C, afterwards inactivation for 20 min at 80 °C. Add 1 µL SAP (shrimp alcaline phosphatase) and 1.2 µL 10 x SAP buffer, incubate for 1 h at 37 °C. Clean up the vector via gel electrophoresis and PCR clean-up kit.
• Ligation: after digestion and clean-up: 50 - 100 ng of vector, 3 - 10 fold molar access of insert, 20 µL ligation volume, 2 µL T4-Ligase-Buffer, 1 µL T4-Ligase. Incubate for 20 - 30 min at room temperature, afterwards inactivation for 5 min at 70 °C. Then: store at -20 °C or transform.

Prefix Insertion

• Digestion of insert: at least 700 ng DNA / 10 µL volume, 1 µL 10x BamHI buffer, 0.5 µL EcoRI, 0.5 µL SpeI. Digest for 2 h at 37 °C, afterwards inactivation for 20 min at 80 °C. Clean up the insert via gel electrophoresis. When cutting the insert out of the gel try to avoid staining or exposure to ultraviolet light of the insert.
• Digestion of vector about 700 ng DNA / 10 µL volume, 1 µL 10 x Tango buffer, 0.5 µL EcoRI, 0.5 µL XbaI. Digest for 2h at 37 °C, afterwards inactivation for 20 min at 80 °C. Add 1 µL SAP (shrimp alkaline phosphatase) and 1.2 µL 10 x SAP buffer, incubate for 1 h at 37 °C. Clean up the vector via gel electrophoresis and PCR clean-up kit.
• Ligation: after digestion and clean-up: 50 - 100 ng of vector, 3 - 10 fold molar access of insert, 20 µL ligation volume, 2 µL T4-Ligase-Buffer, 1 µL T4-Ligase. Incubate for 20 - 30 min at room temperature, afterwards inactivation for 5 min at 70 °C. Then: store at -20 °C or transform.

Variations

• A digestion over night is possible. If you digest over night use only 0.1 µL restriction enzyme.
• It is also possible to use PCR product as insert. Digest after PCR with corresponding restriction enzymes and clean up with PCR clean-up kit. This could lead to higher yields of insert DNA because a lot of DNA gets lost during the gel electrophoresis clean up.

Standard Freiburg BioBrick Assembly

Modified from [http://openwetware.org/wiki/Silver:_BB_Strategy Silver lab] and [http://partsregistry.org/Assembly_standard_25 Assembly standard 25]:

This assembly method can be used for fusion protein assemblies with BioBricks which are bigger than 150 bp. The BioBrick should be at least 500 bp bigger or smaller than the backbone. The BioBrick, which complies with these conditions, is used as the insert and is assembled into the prefix or suffix of the other used BioBrick, which is called vector and needs to be available in the BioBrick [http://partsregistry.org/Assembly_standard_25 Assembly standard 25]. You have to differentiate between a prefix and a suffix insertion.

Suffix Insertion

• Digestion of insert: at least 700 ng DNA / 10 µL volume, 1 µL 10x NEB buffer 4 + 0.1 µL 100x BSA, 0.5 µL NgoMIV (NEB), 1 µL PstI. Digest for 2 h at 37 °C, afterwards inactivation for 20 min at 80 °C. Clean up the insert via gel electrophoresis. When cutting the insert out of the gel try to avoid staining or exposure to ultraviolet light of the insert.
• Digestion of vector about 700 ng DNA / 10 µL volume, 1 µL 10x orange buffer, 0.5 µL AgeI, 0.5 µL PstI. Digest for 2 h at 37 °C, afterwards inactivation for 20 min at 80 °C. Add 1 µL SAP (shrimp alkaline phosphatase) and 1.2 µL 10x SAP buffer, incubate for 1 h at 37 °C. Clean up the vector via gel electrophoresis and PCR clean-up kit.
• Ligation: after digestion and clean-up: 50 - 100 ng of vector, 3 - 10 fold molar access of insert, 20 µL ligation volume, 2 µL T4-Ligase-Buffer, 1 µL T4-Ligase. Incubate for 20 - 30 min at room temperature, afterwards inactivation for 5 min at 70 °C. Then: store at -20 °C or transform.

Prefix Insertion

• Digestion of insert: at least 700 ng DNA / 10 µL volume, 1 µL 10x orange buffer, 0.5 µL EcoRI, 0.5 µL AgeI. Digest for 2 h to 4 h at 37 °C, afterwards inactivation for 20 min at 80 °C. Clean up the insert via gel electrophoresis. When cutting the insert out of the gel try to avoid staining or exposure to ultraviolet light of the insert.
• Digestion of vector about 700 ng DNA / 10 µL volume, 1 µL 10 x NEB buffer 4, 0.5 µL EcoRI, 0.5 µL NgoMIV (NEB). Digest for 2 h to 4 h at 37 °C, afterwards inactivation for 20 min at 80 °C. Add 1 µL SAP (shrimp alcaline phosphatase) and 1.2 µL 10 x SAP buffer, incubate for 1 h at 37 °C. Clean up the vector with a PCR clean-up kit.
• Ligation: after digestion and clean-up: 50 - 100 ng of vector, 3 - 10 fold molar access of insert, 20 µL ligation volume, 2 µL T4-Ligase-Buffer, 1 µL T4-Ligase. Incubate for 20 - 30 min at room temperature, afterwards inactivation for 5 min at 70 °C. Then: store at -20 °C or transform.

Variations

• A digestion over night is possible. If you digest over night use only 0.1 µL restriction enzyme.
• It is also possible to use PCR product as insert. Digest after PCR with corresponding restriction enzymes and clean up with PCR clean-up kit. This could lead to higher yields of insert DNA because a lot of DNA gets lost during the gel electrophoresis clean up.

Restriction analysis

• Digest BioBrick of interest: about 400 ng DNA / 10 µL volume, 1 µL 10x orange buffer, 0.5 µL NotI or PstI. Digest for 2 h at 37 °C. NotI is used to determine the length of the BioBrick and the plasmid backbone, PstI is used to determine the length of the BioBrick in the plasmid backbone.
• Gel electrophoresis: add 2 µL loading buffer to every digestion mix, apply about 100 - 200 ng DNA / pocket in gel. Don't forget to apply the uncut BioBrick as well. A good agarose concentration for BioBricks between 0.2 and 3 kb is 1.5 %. The smaller your BioBrick of interest is the higher the agarose concentration should be and vice versa. The gel electrophoresis is made with TAE-buffer. Be sure that you melt your agarose gel in the same buffer you use for the electrophoresis later.

Colony PCR

• Pick one colony with a sterile tip and elute it in 100 µL ddH₂0 or medium
• Store the colony in 4 °C while colony PCR is running
• One reaction mix contains:
  • 10 µL 5x buffer
  • 2 µL MgCl₂ (25 mM stock)
  • 1 µL dNTPs
  • 0.5 µL primer mix (prefix/suffix primers or sequencing primers)
  • 35.25 µL ddH₂O
  • 0.25 µL GoTaq polymerase (Promega)
  • 1 µL template
• PCR program:
  • Start: 3 min, 98 °C
  • 30 cycles of:
    • 30 s, 98 °C
    • 30 s, 55 °C
    • 30 s / 1 kb template, 72 °C
  • Finish: 5 min, 72 °C

• Gel electrophoresis: check the fragment size
• Plate the correct colonies

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