Colony PCR protocol for Synechococcus elongatus UTEX 2973

1. Pick colony you want to use as template
2. Resuspend in 50µl ddH₂O
3. Boil at 100°C for 10 minutes
4. Afterwards prepare the reaction as follows:

2.5 µl forward primer

2.5 µl reversed primer

25 µl Thermo Scientific™DreamTaq Green PCR Master Mix (2X)

20 µl boiled cells

5. Perform PCR using the following conditions:



step	Temperature	Time	cycle number
Initial Denaturation	95°C	180 s	1
Denaturation	95	30 s	25
Annealing	Tm	30 s	25
Elongation	72	60 s	25
Terminal elongation	72	15 min	1




For PCR products longer than 2kb, the elongation time should be prolonged by 1min/kb.

6. Load your gel

Digestion

1. measurement of concentration of DNA
2. mix components listed in table below




component	volume /µL
DNA (1 µg)	x
H2O	44.0-x
CutSmart Buffer or NEBuffer	5.00
Enzyme e.g. BsmbI or BSaI	1.00
Σ in tube	50.0



3. 2h, 37°C in Mastercycler
4. Agarose gel with Ethidium bromide (EtBr) (3 µL EtBr in 60 mL agarose gel), 70-120 V, 30-60 minutes

DNA plasmid purification with the Macherey-Nagel Kit:

Before starting:

Add 1 mL of Buffer A1 to the RNase A vial and vortex. Transfer the solution back into the Buffer A1 bottle and mix thoroughly. Indicate date of RNase A addition. Store Buffer A1 containing RNase A at 4 °C. The solution will be stable at this temperature for at least six months.

Add the indicated volume of 96-100 % ethanol to Buffer A4 and Buffer AQ.

1. Cultivate and harvest bacterial cells

   Use 1-5 mL of a saturated E.coli LB culture, pellet cells in a standard benchtop microcentrifuge for 30 s at 11,000 x g. Discard the supernatant and remove as much of the liquid as possible.

   Note: For isolation of low-copy plasmids refer to section 5.2.

   ~Personal note: We centrifuged in 15 mL falcons at 4000 rpm for 10 minutes at 4 °C.~



2. Cell lysis

   Add 250 µL Buffer A1. Resuspend the cell pellet completely by vortexing or pipetting up and down. Make sure no cell clumps remain before addition of Buffer A2!

   Attention: Check Buffer A2 for precipitated SDS prior to use. If a white precipitate is visible, warm the buffer several minutes at 30-40 °C until precipitate is dissolved completely. mix thoroughly and cool buffer down to room temperature (18-25 °C).

   Add 250 µL Buffer A2. Mix gently by inverting the tube 6-8 times. Do not vortex to avoid shearing of genomic DNA. incubate at room temperature for up to 5 min or until lysate appears clear.

   Add 300 µL Buffer A3. Mix thoroughly by inverting the tube 6-8 times until blue samples turn colorless completely! Do not vortex to avoid shearing of genomic DNA!

   Make sure to neutralize completely to precipitate all protein and chromosomal DNA. LyseControl should turn completely colorless without any traces of blue.



3. Clarification of lysate

   Centrifuge for 5 min at 11,000 x g at room temperature.

   Repeat this step in case the supernatant is not clear!



4. Wash silica membrane

   Place a NucleoSpin® Plasmid / Plasmid (NoLid) Column in a Collection Tube (2 mL) and decant the supernatant from step 3 or pipette a maximum of 750 µL of the supernatant onto the column. Centrifuge for 1 min at 11,000 x g. Discard flowthrough and place the NucleoSpin® Plasmid / Plasmid (NoLid) Column back into the collection tube.

   Repeat this step to load the remaining lysate.



5. Wash silica membrane

   Recommended: If plasmid DNA is prepared from host strains containing high levels of nucleases (e.g., HB101 or strains of the JM series), it is strongly recommended performing an additional washing step with 500 µL Buffer AW, optionally preheated to 50 °C, and centrifuge for 1 min at 11,000 x g before proceeding with Buffer A4. Additional washing with Buffer AW will also increase the reading length of DNA sequencing reactions and improve the performance of critical enzymatic reactions.

   Add 600 µL Buffer A4 (supplemented with ethanol, see section 3). Centrifuge for 1 min at 11,000 x g. Discard flowthrough and place the NucleoSpin® Plasmid/Plasmid (NoLid) Column back into the empty collection tube.



6. Dry silica membrane

   Centrifuge for 2 min at 11,000 x g and discard the collection tube.

   Note: Residual ethanolic wash buffer might inhibit enzymatic reactions.



7. Elute DNA

   Place the NucleoSpin® Plasmid/Plasmid (NoLid) Column in a 1.5 mL microcentrifuge tube (not provided) and add 50 µL Buffer AE. Incubate for 1 min at room temperature. Centrifuge for 1 min at room temperature. Centrifuge for 1 min at 11,000 x g.

   Note: For more efficient elution procedures and alternative elution buffer (e.g., TE buffer or water) see section 2.5.

   ~Personal note: We heated Buffer AE to 80 °C and used 30-40 µL of it.~

DpnI digest

1. add 2 µL of DpnI to sample
2. incubate at 37°C for 2h
3. Agarosegel with Ethidium bromide (EtBr) (3 µL EtBr in 60 mL agarose gel), 70-120 V, 30-60 minutes

Gel Elektrophoresis

1. Preparation of the buffer and agarose gel



TAE-buffer (50x):




Tris	EDTA-Na2-salt	acetic acid
242.3 g/L	18.6 g/L	60.05 g/L





1.2 % agarose gel:




4.8 g agarose in 400 mL TAE-buffer (1x)



2. On 100 mL 1.2 % Agarose gel 5 µL EtBr are added, stirred, filled into the box and then let it solidify.
3. Add 10 µL of loading dye to 50 µL of the sample.
4. Fill the pockets of the gel: 3 µL of gene ladder in one pocket and 15-20 µL of sample in the other pockets.
5. Apply a voltage of 70-100 V and let gel run for 30-60 minutes.

Gel extraction with the QIAquick® Gel Extraction kit

Notes before starting

• This protocoll is for the purification of up to 10 µg DNA (70 bp to 10 kb).
• The yellow colour of Buffer QG indicates a pH = 7.5. DNA adsorption to the membrane is only efficient at pH ≤ 7.5.
• Add ethanol (96-100%) to Buffer PE before use (see bottle label for volume).
• Isopropanol (100%) and a heating block or water bath at 50°C are required.
• All centrifugation steps are carried out at 17,900 x g (13,000 rpm) in a conventional table-top centrifuge.

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
2. Weigh the gel slice in a colourless tube. Add 3 volumes of Buffer QG to 1 volume gel (100 mg gel ~ 100 µL). The maximum amount of gel per spin column is 400 mg. For > 2% agarose gels, add 6 volumes Buffer QG.
3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). Vortex the tube every 2-3 min to help dissolve gel. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose). If the colour of the mixture is orange or violet, add 10 µL 3 M sodium acetate, pH 5.0, and mix. The mixture turns yellow.
4. Add 1 gel volume isopropanol to the sample and mix.
5. Place a QIAquick spin column in a provided 2 mL collection tube or into a vacuum manifold. To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min or apply vacuum to the manifold until all the samples have passed through the column. Discard the flow-through and place the QIAquick column back into the same tube. For sample volumes of >800 µL, load and spin/ apply vacuum again.
6. If DNA will subsequently be used for sequencing, in vitro transcription, or microinjection, add 500 µL Buffer QG to the QIAquick column and centrifuge for 1 min or apply vacuum. Discard flow-through and place the QIAquick column back into the same tube.

7. To wash, add 750 µL Buffer PE to QIAquick column and centrifuge for 1 min or apply vacuum. Discard flow-through and place the QIAquick column back into the same tube.

   Note: If the DNA will be used for salt-sensitive applications (e.g., sequencing, blunt-ended ligation), let the column stand 2-5 min after addition of Buffer PE.

   Centrifuge the QIAquick column in the provided 2 mL collection tube for 1 min to remove residual wash buffer.

8. Place QIAquick column into a clean 1.5 mL microcentrifuge tube.
9. To elute DNA, add 50 µL Buffer EB (10 mM Tris-Cl, pH 8.5) or water to the centre of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30 µL Buffer EB to the centre of the QIAquick membrane, let the column stand for 1 min, and then centrifuge for 1 min. After the addition of Buffer EB to the QIAquick membrane, increasing the incubation time up to 4 min can increase the yield of purified DNA.
10. If purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.

Golden Gate level 0

1. Mix components listed in table below



component	volume /µL
Entry vector (60 ng/µL)	1.00
TF buffer	1.00
T4 ligase	1.00
BsmbI	1.00
Insert (180 ng/µL)	x
H2O	6.00-x
Σ in tube	10.0



2. Golden Gate program in Mastercycler (Eppendorf)



Note: all the parts, the buffer and enzymes were kept on ice consistently




GoldenGate standard program:
Step 1: 37°C, 2 min
Step 2: 16°C, 5 min
Repetition (30x) of step 1 and 2
Step 3: 60°C, 10 min
Step 4: 80°C, 10 min
Step 5: 4°C, hold until lid is opened

The protocol Golden Gate level 0 can also be found following this link.

Golden Gate level 1

1. Measurement of concentration of parts used
2. Dilution of parts to 20 fmol
3. Preparation of Mastermix (consisting of parts) in PCR-tube (1.00 µL of each part)
4. Addition of 1.00 µL level 1 Ori part
5. Addition of 1.00 µL T4 Lig buffer
6. Addition of 1.00 µL T4 Ligase
7. Addition of 1.00 µL BsaI
8. Pipet-mixing
9. GoldenGate program in Mastercycler (Eppendorf)



Note: all the parts, the buffer and enzymes were kept on ice consistently




GoldenGate standard program:
Step 1: 37°C, 2 min
Step 2: 16°C, 5 min
Repetition (30x) of step 1 and 2
Step 3: 60°C, 10 min
Step 4: 80°C, 10 min
Step 5: 4°C, hold until lid is opened

GoldenGate spaceholder constructs program:
Step 1: 37°C, 2 min
Step 2: 16°C, 5 min
Repetition (50x) of step 1 and 2
Step 3: 4°C, hold until lid is opened

The protocol Golden Gate level 1 can also be found following this link.

Ligation Protocol with T4 DNA Ligase (M0202) (New England BioLabs Inc.)

1. Set up the following reaction in a microcentrifuge tube on ice.(T4 DNA Ligase should be added last. Note that the table shows a ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes.) Use NEBioCalculator to calculate molar ratios



component	20 µL reaction
T4 DNA Ligase Buffer (10X)*	2 µL
Vector DNA (4 kb)	50 ng (0.020 pmol)
Insert DNA (1 kb)	37.5 ng (0.060 pmol)
Nuclease-free water	to 20 μl
T4 DNA Ligase	1 µL




* The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature.



2. Gently mix the reaction by pipetting up and down and microfuge briefly.
3. For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.
4. For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours (alternatively, high concentration T4 DNA Ligase can be used in a 10 minute ligation).
5. Heat inactivate at 65°C for 10 minutes.
6. Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.

PCR Purification with the QIAquick® PCR Purification Kit

Notes before starting

• This protocol is for the purification of up to 10 µg PCR products (100 bp to 10 kb in size).
• Add ethanol (96-100%) to Buffer PE before use (see bottle label for volume).
• All centrifugation steps are carried out at 17.900 x g (13,000 rpm) in a conventional table-top microcentrifuge at room temperature.
• Add 1:250 volume pH indicator I to Buffer PB. The yellow colour of Buffer PB with pH indicator I indicates a pH ≤ 7.5. The adsorption of DNA to the membrane is only efficient at pH ≤ 7.5. If the purified PCR product is to be used in sensitive microarray applications, it may be beneficial to use Buffer PB without the addition of pH indicator I; do not add pH indicator I to buffer aliquots.
• Symbols: ● centrifuge processing; ▴ vacuum processing

1. Add 5 volumes Buffer PB to 1 volume of the PCR reaction and mix. If the colour of the mixture is orange or violet, add 10 µL 3 M sodium acetate, pH 5.0, and mix. The colour of the mixture will turn yellow.
2. Place a QIAquick column in ● a provided 2 mL collection tube or into▴a vacuum manifold. For details on how to set up a vacuum manifold, refer to the QIAquick Spin Handbook.
3. To bind DNA, apply the sample to the QIAquick column and ● centrifuge for 30-60 s or▴apply vacuum to the manifold until all the samples have passed through the column. ● Discard flow-through and place the QIAquick column back in the same tube.
4. To wash, add 750 µL Buffer PE to the QIAquick column ● centrifuge for 30-60 s or▴apply vacuum. ● Discard flow-through and place the QIAquick column back in the same tube.
5. Centrifuge the QIAquick column once more in the provided 2 mL collection tube for 1 min to remove residual wash buffer.
6. Place each QIAquick column in a clean 1.5 mL microcentrifuge tube.
7. To elute DNA, add 50 µL Buffer EB (10 mM Tris-Cl, pH 8.5) or water (pH 7.0-8.5) to the centre of the QIAquick membrane and centrifuge the column for 1 min. For increased DNA concentration, add 30 µL elution buffer to the centre of the QIAquick membrane, let the column stand for 1 min and then centrifuge.
8. If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.

PCR Using Q5® High-Fidelity DNA Polymerase (M0491)

1. Please note that protocols with Q5 High-Fidelity DNA Polymerase may differ from protocols with other polymerases. Conditions recommended below should be used for optimal performance.
   Reaction Setup:
   We recommend assembling all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (98°C). All components should be mixed prior to use. Q5 High-Fidelity DNA Polymerase may be diluted in 1X Q5 Reaction Buffer just prior to use in order to reduce pipetting errors.



component	25 µL reaction	50 µL reaction	final concentration
5X Q5 reaction Buffer	5 µL	10 µL	1X
10 mM dNTPs	0.5 µL	1 µL	200 µM
10 µM Forward Primer	1.25 µL	2.5 µL	0.5 µM
10 µM Reverse Primer	1.25 μl	2.5 µL	0.5 µM
Template DNA	variable	variable	< 1,000 ng
Q5 High-Fidelity DNA Polymerase	0.25 µL	0.5 µL	0.02 U/µL
5X Q5 High GC Enhancer (optional)	(5 µL)	(10 µL)	1X
Nuclease-Free Water	to 25 μl	to 50 µL




Notes: Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary. Overlay the sample with mineral oil if using a PCR machine without a heated lid.
Transfer PCR tubes to a PCR machine and begin thermocycling.
Thermocycling Conditions for a Routine PCR:




step	temp	time
Initial Denaturation	98°C	30 seconds
25-35 cycles	98°C *50-72°C 72°C	5-10 seconds 10-30 seconds 20-30 seconds/kb
Final Extension	72°C	2 minutes
Hold	4-10°C




*Use of the NEBTm Calculator is highly recommended.



2. General Guidelines:
   Template:Use of high quality, purified DNA templates greatly enhances the success of PCR. Recommended amounts of DNA template for a 50 µl reaction are as follows:



DNA	amount
DNA Genomic	1 ng - 1 µg
Plasmid or Viral	1 pg - 10 ng



3. Primers:
   Oligonucleotide primers are generally 20–40 nucleotides in length and ideally have a GC content of 40–60%. Computer programs such as Primer3 can be used to design or analyze primers. The best results are typically seen when using each primer at a final concentration of 0.5 µM in the reaction.
4. Mg⁺⁺ and additives:
   Mg⁺⁺ concentration of 2.0 mM is optimal for most PCR products generated with Q5 High-Fidelity DNA Polymerase. When used at a final concentration of 1X, the Q5 Reaction Buffer provides the optimal Mg⁺⁺ concentration.
   Amplification of some difficult targets, like GC-rich sequences, may be improved by the addition of 1X Q5 High GC Enhancer. The Q5 High GC Enhancer is not a buffer and should not be used alone. It should be added only to reactions with the Q5 Reaction Buffer when other conditions have failed.
5. Deoxynucleotides:
   The final concentration of dNTPs is typically 200 μM of each deoxynucleotide. Q5 High-Fidelity DNA Polymerase cannot incorporate dUTP and is not recommended for use with uracil-containing primers or templates.
6. Q5 High-Fidelity DNA Polymerase concentration:
   We generally recommend using Q5 High-Fidelity DNA Polymerase at a final concentration of 20 units/ml (1.0 unit/50 μl reaction). However, the optimal concentration of Q5 High-Fidelity DNA Polymerase may vary from 10–40 units/ml (0.5–2 units/50 μl reaction) depending on amplicon length and difficulty. Do not exceed 2 units/50 μl reaction, especially for amplicons longer than 5 kb.
7. Buffers:
   The 5X Q5 Reaction Buffer provided with the enzyme is recommended as the first-choice buffer for robust, high-fidelity amplification. For difficult amplicons, such as GC-rich templates or those with secondary structure, the addition of the Q5 High GC Enhancer can improve reaction performance. The 5X Q5 Reaction Buffer is detergent-free and contains 2.0 mM Mg⁺⁺ at the final (1X) concentration.
8. Denaturation:
   An initial denaturation of 30 seconds at 98°C is sufficient for most amplicons from pure DNA templates. Longer denaturation times can be used (up to 3 minutes) for templates that require it.
   During thermocycling, the denaturation step should be kept to a minimum. Typically, a 5–10 second denaturation at 98°C is recommended for most templates.
9. Annealing:
   Optimal annealing temperatures for Q5 High-Fidelity DNA Polymerase tend to be higher than for other PCR polymerases. The NEB Tm Calculator should be used to determine the annealing temperature when using this enzyme. Typically, use a 10–30 second annealing step at 3°C above the T_m of the lower T_m primer. A temperature gradient can also be used to optimize the annealing temperature for each primer pair.
   For high T_m primer pairs, two-step cycling without a separate annealing step can be used (see note 12).
10. Extension:
    The recommended extension temperature is 72°C. Extension times are generally 20–30 seconds per kb for complex, genomic samples, but can be reduced to 10 seconds per kb for simple templates (plasmid, E. coli, etc.) or complex templates < 1 kb. Extension time can be increased to 40 seconds per kb for cDNA or long, complex templates, if necessary.
    A final extension of 2 minutes at 72°C is recommended.
11. Cycle number:
    Generally, 25–35 cycles yield sufficient product. For genomic amplicons, 30-35 cycles are recommended.
12. 2-step PCR:
    When primers with annealing temperatures ≥ 72°C are used, a 2-step thermocycling protocol (combining annealing and extension into one step) is possible.
13. Amplification of long products:
    When amplifying products > 6 kb, it is often helpful to increase the extension time to 40–50 seconds/kb.
14. PCR product:
    The PCR products generated using Q5 High-Fidelity DNA Polymerase have blunt ends. If cloning is the next step, then blunt-end cloning is recommended. If T/A-cloning is preferred, the DNA should be purified prior to A-addition, as Q5 High-Fidelity DNA Polymerase will degrade any overhangs generated.
    Addition of an untemplated -dA can be done with Taq DNA Polymerase (NEB #M0267 ) or Klenow exo^- (NEB #M0212 ).

qPCR

1. Mix the components listed below



component	Volume
template	1 µL
Primer-F	1 µL
Primer-R	1 µL
2x master mix (provided)	10 µL
H2O	7 µL
total volume	20 µL



2. Start program in Mastercycler:

1. 50°C, 2 minutes

2. 2.95°C, 2 minutes

3. 95°C, 15 seconds

4. 60°C, 60 seconds

cycles: 40(cycle 2 to 4)

Quick electroporation of E. coli

This quick electroporation protocol was explained to us by Dr. Alberto Sánchez-Pascuala Jerez from the research group of Prof. Dr. Tobias J. Erb in the Max-Planck-Institute for terrestrial microbiology in Marburg. It was adapted by us and might not be exactly how Dr. Alberto Sánchez-Pascuala Jerez does it.

Optional:

1. Restreak cells from glycerol stock on LB.
2. Inoculate a single colony in liquid LB.

Standard:

1. Inoculate your culture at OD₆₀₀=0.05 from an overnight culture
2. Grow the culture until an OD₆₀₀=0.5 is reached. Note: If the culture reaches OD600= ~0.6 it can still be used, but reinoculating is recommended if higher values are reached.
3. Directly put the culture on ice for 10-15 min.
4. Transfer the culture into a falcon and spin down in a cooled centrifuge at 2500 rpm, 4°C for 10-15 min.Heraeus™ Multifuge™ X1 is used for higher volumes.
5. Wash the cells 2-3 times in dd H2O (or other sterile water).
6. Resuspend in 0.5-2.0 mL water, depending on how many aliquots you want and how big the pellet is.
7. Make 50 µL – 100 µL aliquots.
8. Add DNA to your aliquot on ice.
9. Transfer cell/ DNA mix into an electroporation cuvette on ice. Gene Pulser®/MicroPulser™ Electroporation Cuvettes (green cap) from Bio-Rad are used.
10. Wipe the cuvette with a paper towel to remove any liquid that might cause an arc and place it in the electroporation chamber.
11. Electroporate the sample, directly add recovery medium and transfer the cells into a reaction tube. The following settings are used: 2500 V, 25 µF, 200 Ω, 2 mm. As medium 500 µL SOB medium are added.
12. Incubate the cells at 37°C and 250 rpm for 1h (amp resistance) or 2h (other resistances; kan, cam, spec..)
13. Plate and incubate at 37°C over night.

Sequencing

1. Add Primer, DNA (500-1200 ng) and water
2. Attach sequencing label on eppie
3. Register the label number online on Microsynth website
4. send registered eppies to Microsynth