Team:Bologna/Protocols
From 2008.igem.org
(Difference between revisions)
Line 114: | Line 114: | ||
#* 0.5% big pieces (6-7 Kb) | #* 0.5% big pieces (6-7 Kb) | ||
#* 2-3% small pieces (100 bases) | #* 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. | ||
Revision as of 09:46, 5 August 2008
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 enzyme 1
- 0.5μl of enzyme 2
- 3μl of buffer
- 5μl of DNA
- 20.5μl of H2O(most pure)
- 0.5 μl of BSA
- 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.