From 2009.igem.org

Latest revision as of 17:11, 10 December 2009

Protocols

Plates preparation

1. Autoclave LB medium with 1.5% agar.
2. Cool (at about 50°C, to prevent agar polymerization).
3. Before pouring the plates add antibiotic (Ampicillin 1000x [ ] or Kanamicin 200x [ ]).
4. Put about 20ml of medium per plate.
5. Leave it solidify and store at 4°C.

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Transformation

1. Thaw the competent cells in ice (do not refreeze).
2. Add 0.1-0.3μg of plasmidic DNA or the respective amount of the ligation reaction into microfuge tubes on ice.
3. Add 80μl of cells.
4. Keep on ice for 20min.
5. HeatShock at 42°C for 60sec without agitation.
6. Keep on ice for 2min.
7. Add 0.9 ml of LB medium at room temperature.
8. Incubate at 37°C for 1hr with agitation.
9. Pellet the cells and discard most of supernatant, leaving about 100μl.
10. Resuspend the pellet and streak on plates containing appropriate antibiotics.
11. Incubate the plates overnight at 37°C.

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Inoculation

1. Put 5 ml of LB media in a 15 ml tube.
2. Add the appropriate antibiotic.
3. Pick one colony from the plate with the inoculation loop
4. Immerse the loop into the LB solution.
5. Incubate the plates overnight (12 hours) at 37°C.

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Miniprep

1. Pellet for 10 mins at 4400 rpm and discard most of supernatant.
2. Resuspend pelleted bacterial cells in 250μl of Buffer P1 and tranfer to a microcentrifuge tube.
3. Add 250μl of Buffer P2 and mix thoruoghly by inverting the tube 4-6 times.
4. Add 350μl of Buffer N3 and mix immediately and thoruoghly by inverting the tube 4-6 times.
5. Centrifuge for 10 min at ~18,000 x g in a table-top microcentrifuge.
6. Apply the supernatant (from step 4) to the QIAprep spin column by decanting or pipetting.
7. Centrifuge for 30-60 s. Discard the flow-though.
8. Recommended: Wash the QIAprep spin column by adding 0.5 ml of Buffer PB and centrifuging for 30-60 s. Discard the flow through.
9. Wash QIAprep spin column by adding 0.75ml of Buffer PE and centrifuge for 30-60 s.
10. Discard the flow through and centrifuge for an additional 1 min to remove residual wash buffer.
11. To elute DNA, place the QIAprep column in a clean 1.5 ml microcentrifuge tube. Add 30μl of Buffer EB (or water) to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.

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Digestion reaction

E/S cut

enzyme 1 --> ECO R1

enzyme 2 --> SPE

buffer --> ECO R1

X/P cut

enzyme 1 --> Xba

enzyme 2 --> Pst 1

buffer --> 3

S/P cut

enzyme 1 --> SPE

enzyme 2 --> Pst 1

buffer --> 2

E/P cut

enzyme 1 --> ECO R1

enzyme 2 --> Pst 1

buffer --> ECO R1

1. Mix:
   • 0.5 μl of BSA
   • 0.5μl of each enzyme
   • 3μl of buffer
   • 5μl of DNA
   • 20.5μl of H2O(most pure)
2. Mix and then spin down.
3. 1h at 37 °C.
4. 20 min at 80 °C to inactivate rescriction enzymes.
5. Put in ice.
6. Start gel run preparation.

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Gel preparation

1. 150ml of Buffer TBE 1x.
2. Add the appropriate quantity of Agarose for the desired thickness
   • 0.7%= 1g of agarose
   • 0.7-1% ( from 400 bases to 3Kb DNA fragments)
   • 0.5% (6-7 Kb fragments)
   • 2-3% (50-100 base long fragments)
3. Microwaves for 2 min.
4. Cool down under flowing water.
5. Add 10μl of EtBr.
6. Prepare the gel tray and set in place the wide-thoot comb.
7. Pour the gel in the gel tray (work in the hood for safety purpose)
8. Get rid of bubbles and let solidify.

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Gel Electrophoresis

1. Put the solidified gel in the apposite run container.
2. Check that TBE fully covers the gel.
3. Gently extract the comb.
4. Add the Loading buffer in the digested DNA (6 ul of loading 6X in 30 ul of digestion volume).
   
5. Make the deposit in the wells of the samples and loading of reference (a part of loading should be prepared to scale by reference).
6. Close the container.
7. Start the run:
   • 50/100 V --> until separate bands
   • 120 V --> until half run
   • 140 V--> until end of run.

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Gel extraction

1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
2. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg ~ 100 μl).
3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). To help dissolve gel, mix by vortexing the tube every 2-3 min during the incubation.
4. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose).
5. Add 1 gel volume of isopropanol to the sample and mix.
6. Place a QIAquick spin column in a provided 2 ml collection tube or into a vacuum manifold.
7. To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min or apply vacuum to the manifold until all samples have passed through the column. Discard flow-through and place the QIAquick column back into the same tube.
8. Recommended: add 0.5 ml of Buffer QG to QIAquick column and centrifuge for 1 min or apply vacuum. Discard flow-through and place the QIAquick column back into the same tube.
9. To wash, add 0.75 ml of 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.
10. Centrifuge the column in a 2 ml collection tube (provided) for 1 min at 18,000 x g.
11. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
12. To elute DNA, add 30 μl of Buffer EB (10 mM Tris-Cl, pH 8.5) or water to the center of the QIAquick membrane and centrifuge the column for 1 min. Alternatively, for increased DNA concentration, add 30 μl of elution buffer to the center of the QIAquick membrane, let the column stand for 1 min and then centrifuge for 1 min.
13. 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.

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Ligation reaction

1. Ligation with one insert and one vector --> f.v.=20μl
   • 8μl of insert
   • 4μl of vector
   • 1μl of T4 ligase
   • 4μl of Buffer 5X
   • 3μl of H2O mQ
2. Ligation with two insert and one vector --> f.v.=30μl
   • 4μl of vector
   • 8μl of insert 1
   • 8μl of insert 2
   • 1μl of T4 ligase
   • 6μl of Buffer 5X
   • 3μl of H2O mQ
3. Conservation:
   • 40 min at Room Temperature, or
   • 3 hours at 15°C

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Chemiocompetent cells

1. Take some colonies of DH5α cells from a fresh streaked plate and inoculate into 125ml of Soc medium.
2. Grow the cells overnight at 25°C (it is advised to grow them slowly in order to have them better synchronized). It takes approximately 20 hours. It is advisable to grow the cells at 25°C overnight and then to shift them at 37°C. Bacteria are ready for harvesting when OD600 is between 0.37 and 0.4. Higher OD will lead to less competent cells (it is important to harvest the bacteria when they are still in the logarithmic phase of growth).
3. transfer the culture into 50 ml falcon tubes.
4. Centrifuge at maximum speed at 4°C for 10 min.
5. Re-dissolve all the cells in 40ml of Transformation buffer.
6. Incubate on ice for 10 min.
7. Spin down the cells (at maximum speed) at 4°C for 10 min.
8. Re-dissolve the pellet in 10ml of Transformation buffer.
9. Add 700μl of DMSO.
10. Aliquot (250μl) and freeze at -80°C.

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Mediums and buffers

Soc medium

1. To prepare 200 ml of SOC medium dissolve in ultrapure water:
   • Tryptone 4g
   • Yeast extract 1g
   • 1N NaCl 2ml
   • 1N KCl 0.5ml
   • 5M NaOH 40 μl to adjust the pH to 6.8
2. After autoclave add 2ml each of 2M Mg-salt (1M MgSO4 and 1M MgCl) and 2M glucose.

LB medium

1. To prepare 1L of LB medium dissolve in ultrapure water:
   • Tryptone 10g
   • Yeast extract 5g
   • NaCl 10g
   • 5M NaOH 200μl
2. Autoclave and store at room temperature.

Transformation Buffer

(to be always made fresh)

1. To prepare 60 ml dissolve in ultrapure water:
   • (15mM) CaCl2 0.13g
   • (250mM) KCl 1.12g
   • (10mM) Pipes 0.18g
2. Adjust pH with KOH to 6.7.
3. Add (55mM) MnCl2 0.65g.
4. Filter with 0.22μm filter.

M9 medium

1. Dissolve 56.4g in 1l of distilled water.
2. Autoclave for 15 min at 121°C.

This convenient 5x concentrate can be stored and diluted as needed to prepare 5l of 1x M9 minimal salts. For M9 medium:

1. Aseptically mix 200ml of 5X M9 minimal salts 5x[ ] with 643.9 ml of sterile water, if necessary, cool to 45-50°C.
2. Aseptically add 20ml of sterile 1M glucose and 2mL of sterile 1M magnesium solfate,0.1ml of 1M sterile calcium chloride, 34 ml Thyamine Hydroclhoride and 100 ml 2% casa-amino acids.

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Antibiotics stocks preparation

Ampicillin

1. Dissolve ampicillin in ultrapure water 100mg/mL (stock 1000x [ ], working concentration 100 μg/mL).
2. Aliquot and store at -20°C.

Kanamycin

1. Dissolve Kanamicin in ultrapure water 50mg/mL (stock 1000x [ ], working concentration 50μg/mL).
2. Aliquot and store at -20°C.

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IPTG stocks preparation

1. IPTG (Isopropyl β-D-1-thiogalactopyranoside)0.1 M stocks.

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Fluorescence test

1. Growth O/N for 12h in M9 medium (5ml) of:
   • transformed E. coli with appropriate antibiotic;
   • In the morning, measure OD.
   • centrifuge 1ml of bacterial culture at 4400rpm for 3min at 25°C;
   • discard the supernatant;
   • resuspend the cell pellet in some microliters of M9 medium.
   • slide preparation and image analisis.
2. IPTG induction:
   • centrifugate bacteria culture at 4400rpm for 3min at 25°C;
   • discard the supernatant;
   • resuspend cell pellet in M9 medium and IPTG with appropriate antibiotic (1000x [ ]);
3. Incubate at 37°C.
4. Test fluorescence after the chosen time intervals.

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M9 supplemented media

For 1L of 1X media

• 200 mL 5X M9 minimal salts
  1. Dissolve 56.4 g Bacto M9 minimal salts, 5X from Difco in 1L H2O
  2. Separate into 200 mL aliquots
  3. Autoclave to sterilize. 121°C for 15 minutes.
• 34 mL 10 mg/mL thiamine
  1. Dissolve 10 mg per mL of H2O
  2. Use a 0.22 μm filter to filter-sterilize
• 10 mL 40% glycerol
  1. Add 80 mL glyerol to 120 mL of H2O
  2. Autoclave to sterilize
• 20 mL 10% Casamino acids
  1. Dissolve 50 g Bacto Casamino acids from Difco in 500 mL H2O
  2. Autoclave to sterilize
• 2 mL 1M MgSO4
  1. Dissolve 24.65 g MgSO4·7H2O in 100 mL H2O
  2. Autoclave to sterilize
• 100 μL 1M CaCl2
  1. Dissolve 14.7 g CaCl2·2H2O in 100 mL H2O
  2. Autoclave to sterilize
• 733.9 mL H2O
  1. Sterilize deionized water in autoclave

Combine above solutions using sterile technique. (May notice some precipitation during preparation but precipitate should go back into solution once volume is brought up to 1L with sterile H2O.) Add antibiotic as appropriate and store at 4°C

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