Team:Bologna/Protocols

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HOME THE PROJECT THE TEAM PARTS SUBMITTED TO THE REGISTRY MODELING NOTEBOOK


Contents

Plates preparation

  1. Autoclave LB medium with 2% agar.
  2. Cool at 50°C to prevent agar polymerization.
  3. Before preparing 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.


Biobricks amplification

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


Transformation

  1. Thaw the competent cells in ice (do not refreeze).
  2. Dispense 100μl 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. Heat at 42°C for 60sec without agitation.
  6. Keep on ice for 2min.
  7. Add 0.9ml 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.


Inoculation

  1. Put 5 ml of LB in a falcon tube.
  2. Add the appropriate antibiotic.
  3. Pick one spot of cells from the colony with "ansa"
  4. Put cells in solution.
  5. Incubate the plates overnight (12 hours) at 37°C.
  6. Pellet for 10 mins at 4400 rpm and discard most of supernatant.


Miniprep

  1. Resuspend pelleted bacterial cells in 250μl Buffer P1 and tranfer to a microcentrifuge tube.
  2. Add 250μl Buffer P2 and mix thoruoghly by inverting the tube 4-6 times.
  3. Add 350μl Buffer N3 and mix immediately and thoruoghly by inverting the tube 4-6 times.
  4. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
  5. Apply the supernatant (from step 4) to the QIAprep spin column by decanting or pipetting.
  6. Centrifuge for 30-60 s. Discard the flow-though.
  7. Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30-60 s. Discard the flow through.
  8. Wash QIAprep spin column by eding 0.75ml Buffer PE and centrifuge for 30-60 s.
  9. Discard the flow through and centrifuge for an additional 1 min to remove residual wash buffer.
  10. 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.


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. 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.


Gel preparation

  1. 150ml of Buffer TBE 1x.
  2. Add the appropriate quantity of Agarose.
    • 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. Put in microwaves for 2 min.
  4. Cool down under water.
  5. Add 10μl of EtBr.
  6. Prepare the gel room and big comb.
  7. Put down all in the gel room under the hood.
  8. Discard the bubbles and let solidify.


Electrophoretic run

  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.


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 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.


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 Tamb, or
    • 3 hours at 15°C


Chemiocompetent cells

  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 (200 μl) and freeze at -80°C.


Mediums and buffers

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


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