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Revision as of 10:38, 28 October 2008 (view source) Mavi.amaduzzi (Talk | contribs) (→M9 supplemented media) ← Older edit		Latest revision as of 13:45, 28 October 2008 (view source) Mavi.amaduzzi (Talk | contribs) (→Mediums and buffers)
Line 288:		Line 288:
	; Transformation Buffer		; Transformation Buffer
-	(always made fresh)	+	(to be always made fresh)
	# To prepare 100ml dissolve in ultrapure water:		# To prepare 100ml dissolve in ultrapure water:
	#* 15mM CaCl2 0.2205g		#* 15mM CaCl2 0.2205g

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Latest revision as of 13:45, 28 October 2008

HOME	TEAM	PROJECT	MODELING	WET-LAB	SOFTWARE	SUBMITTED PARTS	BIOSAFETY AND PROTOCOLS

Contents 1 Plates preparation 2 Biobricks amplification 3 Transformation 4 Inoculation 5 Miniprep 6 Digestion reaction 7 Gel preparation 8 Electrophoretic run 9 Gel extraction 10 Ligation reaction 11 Chemiocompetent cells 12 Mediums and buffers 13 Antibiotics stocks preparation 14 IPTG stocks preparation 15 Fluorescence test 16 M9 supplemented media

Plates preparation

Biosafety level: 1

1. Autoclave LB medium with 2% agar.
2. Cool at 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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Biobricks amplification

Biosafety level: 1

1. Puncture a hole through the foil with a pipette tip (wash it everytime with bleach-distilled water-EOH 95%) into the spot that corresponds to the Biobrick™-standard part that you want.
2. Soak the paper in 5μl of TE buffer.
3. Rest for 20 minutes at 50°C.

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Transformation

Biosafety level: 1

1. Thraw the competent cells in ice (do not refreeze).
2. Dispense 80μl of cells into microfuge tubes on ice.
3. Add 0.1-0.3μg of plasmidic DNA or the respective amount of the ligation reaction.
4. Keep on ice for 30min.
5. HeatShock at 42°C for 60sec without agitation.
6. Keep on ice for 2min.
7. Add 0.8ml 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. Streak on plates containing appropriate antibiotics.
11. Incubate the plates overnight at 37°C.

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Inoculation

Biosafety level: 1

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

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Miniprep

Biosafety level: 1

1. Pellet for 10 mins at 4400 rpm and discard most of supernatant.
2. Resuspend pelleted bacterial cells in 250μl Buffer P1 and tranfer to a microcentrifuge tube.
3. Add 250μl Buffer P2 and mix thoruoghly by inverting the tube 4-6 times.
4. Add 350μl Buffer N3 and mix immediately and thoruoghly by inverting the tube 4-6 times.
5. Centrifuge for 10 min at 13,000 rpm (~17,900 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 Buffer PB and centrifuging for 30-60 s. Discard the flow through.
9. Wash QIAprep spin column by eding 0.75ml 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 elude DNA, place the QIAprep column in a clean 1.5 ml microcewntrifuge tube. Add 30μl Buffer EB or water to the center of each QIAprep spin solumn, let stand for 1 min, and centrifuge for 1 min.

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Digestion reaction

Biosafety level: 1

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. Spinning down.
3. 1h at 37 °C.
4. 20 min at 80 °C to block the enzymatic action.
5. Put in ice.
6. Start run preparation.

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Gel preparation

Biosafety level: 2

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% general( from 200 bases to 3Kb)
   • 0.5% big pieces (6-7 Kb)
   • 2-3% small pieces (100 bases)
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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Electrophoretic run

Biosafety level: 2

1. Take the room with solidified gel and put it in the apposite run container.
2. Check that all liquid (TBE) cover fully the gel, otherwise add the necessary liquid.
3. Decrease gently the comb.
4. Add the Laoading in the digested DNA.
   

Loading: substance that loads the sample with dye so that we can see the evolution of race and weight the DNA so that deposits in the well; concentrated at 6x.

1. Make the deposit in the wells of the samples and loading of reference (a part of loading should be prepared to scale by reference).
2. Close the container.
3. Start the run:
   • 50/100 V --> until separete bands
   • 120 V --> until half run
   • 140 V--> until end of run.

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Gel extraction

Biosafety level: 2

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 ina 2 ml collection tube (provided) for 1 min at 17,900 x g (13,000 rpm).
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 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

Biosafety level: 1

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 Tamb, or
   • 3 hours at 15°C

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Chemiocompetent cells

Biosafety level: 1

1. Take some colonies of DH5α cells from a fresh streaked plate and inoculate 125ml of Soc medium into a 2l flask.
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. Spin down the cells (at maximum speed) at 4°C for 10 min.
4. Re-dissolve the pellet in 40ml of Transformation buffer.
5. Incubate on ice for 10 min.
6. Spin down the cells (at maximum speed) at 4°C for 10 min.
7. Re-dissolve the pellet in 10ml of Transformation buffer.
8. Add 700μl of DMSO.
9. Aliquot (200μl) and freeze at -80°C.

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Mediums and buffers

Biosafety level: 1

Soc medium

1. To prepare 1l of SOC medium dissolve in ultrapure water:
   • Tryptone 4g
   • Yeast extract 1g
   • 1M NaCl 2ml
   • 1M KCl 0.5ml
   • 5M NaOH 200μl to adjust the pH to 6.8
2. After autoclaving 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 100ml dissolve in ultrapure water:
   • 15mM CaCl2 0.2205g
   • 250mM KCl 1.864g
   • 10mM Pipes 0.302g
2. Adjust pH with KOH to 6.7.
3. Add 55mM MnCl2 (0.89g of MnCl2x2H2O).
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 minimal medium:

1. Aseptically dilute 200ml of M9 minimal salts, 5x[ ] with 800mL 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 to prepare 1l of M9 minimal medium.
3. If desired aseptically add 0.1ml of 1M sterile calcium chloride to the M9 minimal medium. M9 minimal medium may also be supplemented with the appropriate amino acids.

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Antibiotics stocks preparation

Biosafety level: 1

Ampicillin

1. Dissolve ampicillin in ultrapure water 100mg/mL (stock 1000x [ ], working concentration100 μg/mL).
2. Aliquot and store at -20°C.

Kanamicin

1. Dissolve Kanamicin in ultrapure water 10mg/mL (stock 200x [ ], working concentration 50μg/mL).
2. Aliquot and store at -20°C.

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IPTG stocks preparation

Biosafety level: 1

1. IPTG (Isopropyl β-D-1-thiogalactopyranoside) 100mM stocks preparation for Plac induction (IPTG MW=238.31):
   • 0.476g in 20ml of ultrapure water. Working concentration 1mM

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Fluorescence test

Biosafety level: 1

1. Growth O/N for 15h in LB (5ml) of:
   • transformed E. coli with appropriate antibiotic;
   • E. coli.
2. All measures must be done in M9 with OD=1.2.
3. The day after, in the morning, measure OD.
4. To obtain OD=1.2:
   • centrifuge 4ml of bacterial culture at 4400rpm for 3min at 25°C;
   • discard the supernatant;
   • resuspend the cell pellet in 6ml of M9 medium with glucose and appropriate antibiotic (1000x [ ]).
5. Measure OD: adjust OD to 1.2 through further dilution or cell growth.
6. Adjust PMT offset with untransformed E. Coli.
7. Test culture fluorescence before IPTG induction.
8. IPTG induction:
   • centrifugate bacteria culture at 4400rpm for 3min at 25°C;
   • discard the supernatant;
   • resuspend cell pellet in M9 medium and 2mM IPTG with appropriate antibiotic (1000x [ ]);
9. Incubate at 37°C.
10. Test fluorescence after 10min, 20min, 30min, 1h, 2h.

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M9 supplemented media

Biosafety level: 1

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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