Team:Bologna/Protocols
From 2008.igem.org
Revision as of 09:10, 5 August 2008 by Mavi.amaduzzi (Talk | contribs)
HOME | THE PROJECT | THE TEAM | PARTS SUBMITTED TO THE REGISTRY | MODELING | NOTEBOOK |
---|
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 enzyme 1
- 0.5μl of enzyme 2
- 3μl of buffer
- 5μl of DNA
- 20.5μl of H2O(most pure)
- 0.5 μl of BSA
- Quick centrifuge.