Team:Bologna/Protocols
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HOME | THE PROJECT | THE TEAM | PARTS SUBMITTED TO THE REGISTRY | MODELING | NOTEBOOK |
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Contents |
Plates preparation
- Autoclave LB medium with 2% agar.
- Cool at 50°C to prevent agar polymerization.
- Before preparing plates add antibiotic (Ampicillin 1000x or Kanamicin 200x).
- Put about 20ml of medium per plate.
- Leave it solidify and store at 4°C.
Biobricks amplification
- 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.
- Add 5μl of TE buffer.
- Rest for 20 minutes at 50°C.
Transformation
- Thaw the competent cells in ice (do not refreeze).
- Dispense 100μl of cells into microfuge tubes on ice.
- Add 0.1-0.3μg of plasmidic DNA or the respective amount of the ligation reaction.
- Keep on ice for 30min.
- Heat at 42°C for 60sec without agitation.
- Keep on ice for 2min.
- Add 0.9ml of LB medium at room temperature.
- Incubate at 37°C for 1hr with agitation.
- Pellet the cells and discard most of supernatant, leaving about 100μl.
- Streak on plates containing appropriate antibiotics.
- Incubate the plates overnight at 37°C.
Inoculation
- Put 5 ml of LB in a falcon tube.
- Add the appropriate antibiotic.
- Pick one spot of cells from the colony with "ansa"
- Put cells in solution.
- Incubate the plates overnight (12 hours) at 37°C.
- Pellet for 10 mins at 4400 rpm and discard most of supernatant.
Miniprep
- Resuspend pelleted bacterial cells in 250μl Buffer P1 and tranfer to a microcentrifuge tube.
- Add 250μl Buffer P2 and mix thoruoghly by inverting the tube 4-6 times.
- Add 350μl Buffer N3 and mix immediately and thoruoghly by inverting the tube 4-6 times.
- Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
- Apply the supernatant (from step 4) to the QIAprep spin column by decanting or pipetting.
- Centrifuge for 30-60 s. Discard the flow-though.
- Recommended: Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30-60 s. Discard the flow through.
- Wash QIAprep spin column by eding 0.75ml Buffer PE and centrifuge for 30-60 s.
- Discard the flow through and centrifuge for an additional 1 min to remove residual wash buffer.
- 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
- Mix:
- 0.5 μl of BSA
- 0.5μl of each enzyme
- 3μl of buffer
- 5μl of DNA
- 20.5μl of H2O(most pure)
- Spinning down.
- 1h at 37 °C.
- 20 min at 80 °C to block the enzymatic action.
- Put in ice.
- Start run preparation.
Gel preparation
- 150ml of Buffer TBE 1x.
- 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)
- Put in microwaves for 2 min.
- Cool down under water.
- Add 10μl of EtBr.
- Prepare the gel room and big comb.
- Put down all in the gel room under the hood.
- Discard the bubbles and let solidify.
Electrophoretic run
- Take the room with solidified gel and put it in the apposite run container.
- Check that all liquid (TBE) cover fully the gel, otherwise add the necessary liquid.
- Decrease gently the comb.
- 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.
- Make the deposit in the wells of the samples and loading of reference (a part of loading should be prepared to scale by reference).
- Close the container.
- Start the run:
- 50/100 V --> until separete bands
- 120 V --> until half run
- 140 V--> until end of run.
Gel extraction
- Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.
- Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (100 mg ~ 100 μl).
- 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.
- After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose).
- Add 1 gel volume of isopropanol to the sample and mix.
- 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 samples have passed through the column. Discard flow-through and place the QIAquick column back into the same tube.
- 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.
- 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.
- Centrifuge the column ina 2 ml collection tube (provided) for 1 min at 17,900 x g (13,000 rpm).
- Place QIAquick column into a clean 1.5 ml microcentrifuge tube.
- 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.
- 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
- 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
- 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
- Conservation:
- 40 min at Tamb, or
- 3 hours at 15°C