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Protocols

Transforming One Shot® Mach1™ competent cells
Mini-prep plasmid isolation for E. coli and S. cerevisae
Freezing cell stocks
Restriction enzyme digestion
Ligation
Running DNA Gel
PCR

Transforming One Shot® Mach1™ competent cells

Materials:

• - Competent cells
• - SOC medium (warmed to room temperature)
• - Plasmid DNA or DNA ligation mix
• - LB agar plates containing 15-100 g/mL antibiotic of choice, pre-warmed to 37 °C
• - water bath at 42 °C
• - shaking incubator at 37 °C.

Protocol:

1. Add 50-100 ng DNA into a 20 L competent E.coli, and mix gently. Do not mix by pipetting up and down!
2. Incubate tube vial on ice for 30 minutes
3. Heat-shocks the cells for 30 seconds at 42 °C without shaking
4. Immediately transfer the tubes back to ice for 2 minutes
5. Add 250 L of room temperature LB medium
6. Cap tube tightly and shake tube horizontally (225 rpm) at 37 °C for 1 hour
7. Plate from each tube 100 L on an agar plate containing antibiotic. Spin tube, discard supernatant to leave no more than 100 L, vortex and plate on an agar plate
8. Incubate plates overnight at 37 °C

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Qiagen Mini-prep plasmid isolation for E. coli and S. cerevisae

This protocol is based on QIAGEN® Plasmid Purification Handbook.
Materials:

• - bacterial or yeast culture
• - Qiagen colums
• - buffer P1 (100 mg/mL RNAse A, 50 mM Tris/HCl, 10 mM EDTA, pH 8.0)
• - buffer P2 (200 mM NaOH, 1% SDS)
• - buffer P3 (3 M KAc, pH 5.5)
• - buffer PE
• - milliQ pH 8.0
• - centrifuge
• - nanodrop
• - for yeast plasmid isolation: zymolyase 5 U/μl

Protocol:

1. Pick a single colony from a freshly streaked selective plate and inoculate a starter culture of 2–5 mL LB medium containing the appropriate selective antibiotic or selective medium. Incubate for approximately 8 h at 37°C (bacteria) or 12 h at 30°C with vigorous shaking (approx. 300 rpm)
2. Harvest the 5 mL bacterial cells by centrifugation at 13,000 rpm for 1 min at 20°C (microcentrifuge tube). If you wish to stop the protocol and continue later, freeze the cell pellets at –20°C
3. For bacteria: Resuspend pelleted bacterial cells in 250 μL Buffer P1. Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet.
   For yeast: Resuspend cells in 250 μL Buffer P1 with 3 μL zymolyase and incubate 1 h at 37 °C
4. Add 250 μL Buffer P2 and mix thoroughly by inverting the tube 4–6 times. Mix gently by inverting the tube. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 minutes.
5. Add 350 μL Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. To avoid localized precipitation, mix the solution thoroughly, immediately after addition of Buffer N3. Large culture volumes (e.g. ≥5 mL) may require inverting up to 10 times. The solution should become cloudy.
6. Incubate at -20 °C for 15 minutes.
7. Centrifuge for 10 min at 13,000 rpm in a table-top microcentrifuge. A compact white pellet will form.
8. Apply the supernatants from step 7 to the QIAprep spin column by decanting or pipetting.
9. Centrifuge for 30–60 seconds. Discard the flow-through.
10. Wash QIAprep spin column by adding 0.75 mL Buffer PE and centrifuging for 30–60 seconds.
11. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer. Important: Residual wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions.
12. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 30 μL Buffer EB (10 mM Tris•Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 minute in 50 C stove and centrifuge for 1 minute to obtain DNA.
13. Measure DNA concentration on the Nanodrop

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Freezing cell stocks

Materials:

• - bacterial culture
• - Growth medium
• - 80% glycerol
• - centrifuge

Protocol:

1. Take 5 mL bacterial cells from the Erlenmeyer of a freshly grown culture and spin in a 15 mL tube for 10 minutes at 2.000 rpm (Eppendorf centrifuge)
2. Decant the supernatant without disturbing the pellet
3. Pipet on the pellet 0.5 ml of appropriate medium and 0.5 mL 80% glycerol and mix by vortexing and save in -80 °C freezer

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Restriction enzyme digestion

Materials:

• - plasmid DNA or PCR product
• - restriction enzymes (Roche and BioLabs)
• - buffer (10x)
• - H2O
• water bath at 37 °C
• - heat block or water bath at 65 °C

Protocol:
Digestions (cutting plasmid DNA) were performed at the appropriate temperature with the appropriate buffer in the appropriate concentration, according to the supplier. With double restriction, use bigger volume (~ 50 μL) and we found out that adding BSA altered performance greatly.

Reaction for one sample:
DNA × μL (up to 1,0 μg)
Buffer (10×) × μL (1×))
Restriction enzymes × μL (5 units/μg DNA = 1 µL) )
H2O × μL
tot volume 20-25 μL

Incubate for (at least) one hour at 37 °C. Inactivate the restriction endonucleases by heat, incubation at 65 °C for 10 minutes and centrifuge shortly.
Used Buffers:
Buffer H (Roche): 50 mM Tris-HCl, 1 M NaCl, 100 mM MgCl2, 10 mM DTE, pH 7.5 at 37 °C
Buffer M (Roche): 100 mM Tris-HCl, 500 mM NaCI, 100 mM MgCl2, 10 mM DTE, pH 7.5 at 37 °C
Buffer 1 (BioLabs): 10 mM Bis-Tris-Propane-HCl, 10 mM MgCl2, 1 mM DTE,pH 7.0 at 25°C
Buffer 2 (BioLabs): 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM DTE, pH 7.9 at 25°C
Buffer 3 (BioLabs): 100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl2, 1 mM DTE, pH 7.9 at 25°C
Buffer 4 (BioLabs): 50 mM CH3CO2K, 20 mM TAE, 10 mM Mg(CH3COO)2, 1 mM DTE, pH 7.9 at 25°C

Note: some of the restriction enzymes of New England BioLabs required the addition of 100 µg/mL BSA)

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Ligation

Materials:

• - digested plasmid DNA or PCR product
• - T4 ligation buffer (10x) (Fermentas)
• - T4 ligase (Fermentas)
• - H2O
• water bath at 16 °C

Protocol: Ligations (pasting plasmid DNA) were performed at the appropriate temperature with the appropriate buffer in the appropriate concentration, according to the supplier. We encountered different tactics for ligation. Usually it comes down to keeping it ~16 °C for at least 3 hours.
Reaction for one sample:
DNA insert × μL
DNA vector × μL
T4 Ligation buffer (10×) × μL (for 1×)
T4 Ligase 1.0 μL
H2O × μL
tot. volume 10-15 μL

The final concentration is preferably ~100 ng/μL. Smaller volumes are preferred and when DNA is at low concentration, try to evaporate water using a vacuum. Incubate at 16 °C for at least 3 hours or keep in an ice box in a floatie overnight. In the morning you find eppendorf tubes floating in water which had a temperature gradient overnight. For transformation use circa half of the ligation mix.

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Running a DNA gel

Materials:

• - Agarose
• - TAE 1x
• - SybrSafe DNA stain
• - Loading Dye
• - DNA ladder (Smartladder)
• - DNA electrophoresis machine

Protocol:

1. Dissolve agarose (w/v 0.6% for separating long DNA pieces (>10 kbp), 1% for separating shorter pieces) in 1x TAE by microwaving
2. Close sides of electrophoresis tray (scotch tape works fine) and add comb
3. Let solution cool and add 5 μl Sybrsafe to an empty electrophoresis tray (small gels) 10-12 μl Sybrsafe for larger gels
4. Pour gel until a height of ~0.5 cm. Mix and remove bubbles with pipet tip (fast! It hardens quickly)
5. Put tray into electrophoresis casing and add TAE until a small layer above the gel can be seen. Remove comb
6. Add 1 μl loading dye to 5 μl sample, mix and load in the gel. Also add 5 μl smartladder for your reference
7. Run gel on 80 V (long run)- 110 V (short run, mostly for a ‘fast check’) for ~40-60 minutes, dependant of gel size, separation acquisition and voltage.

For information about the smartladder, Smartladder specifications.

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PCR

Materials:
For PCR with minimal errors, pFX polymerase is used:

• Pfx polymerase (Invitrogen)
• 10x Pfx Buffer (Invitrogen)
• enhancer (Invitrogen)
• 50 mM MgSO4 (Invitrogen)
• 10 mM dNTPs

For PCR for checks of length, Taq polymerase is used, provided in Qiagen mastermix:

• Mastermix

Always needed:

• primer solutions 5 mol/mL
• template DNA (plasmid at 50 pg – 1 ng/μL), or plate with colonies
• PCR machine


Protocol:
First make sure that there is a PCR machine available for you. Take the solutions from the freezer and thaw them on ice.

Preparation of reaction mixture:
1. Gently vortex and briefly centrifuge all solutions after thawing
2. Keep solutions on ice
3. Add to a thin walled PCR tube, on ice the desired reaction mixture listed below.
4. for PCR on colonies: prick a sterile toothpick into a colony, dip it into a PCR tube and put it in 15 mL culture tube containing growth media to grow overnight for direct culturing positives.
5. Make sure you keep everything cool until it enters the preheated PCR machine

For Pfx PCR the reaction mixture is:

Component	Sample
10 mM dNTPs	1.5 μL
Enhancer (Invitrogen)	5.0 μL
10x Pfx Buffer (Invitrogen)	5.0 μL
50 mM MgSO4 (Invitrogen)	1.0 μL
Primer 1	3.0 μL
Primer 2	3.0 μL
Pfx polymerase (Invitrogen)	0.6 μL
DNA template	1.0 μL
H2O	29.9 μL

 

PCR program pFX:

Step	Annealing Temperature	Time, min:sec	Number of cycles
Initial denaturation	95 °C	2:00	1
Annealing	X °C *	1:00
Extension	68°C	1:00-2:00 (1 min/kb)
Denaturation	95 °C	1:00	25
Annealing	X °C *	1:00
Extension	68 °C	1:00-2:00 (1 min/kb)
Final Extension	68 °C	10:00	1

 

For Taq PCR the reaction mixture is:

Component	Sample
Taq PCR Master Mix (Qiagen	12.5 μL
Primer 1	1.5 μL
Primer 2	1.5 μL
Template DNA (or pick a colony)	1 μL
H2O	8.5 μL

 

PCR program Taq polymerase:

Step	Annealing Temperature	Time, min:sec	Number of cycles
Initial denaturation	94 °C	10:00	1
Annealing	X °C *	0:45
Extension	72°C	1:00-2:00 (1 min/kb)
Denaturation	95 °C	1:00	25
Annealing	X °C *	1:00
Extension	72 °C	1:00-2:00 (1 min/kb)
Final Extension	72 °C	10:00	1

 

 




*Annealing temperature is very dependent on primer. Optimal temperature: 3x G/C + 2x A/T