Team:Colombia/Notebook/Protocols

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Protocols

Bacterial (Xam) DNA extraction protocol

1. Shaking culture overnight.
2. Centrifuge twice at 10,700 rpm in 1.5 mL tube 2 or 3 minutes. On the same tube centrifuge all the cultures and remove the supernatant.
3. Wash the pellet twice with 1 mL NaCl 1 M and Resuspend.
4. Centrifuge 2 minutes at 10,700 rpm and remove the supernatant.
5. Resuspend the pellet on 567 μL TE (10 mM Tris-HCl pH 8; 1 mM EDTA pH 8).
6. Add 3 μL proteinase K (20 mg/mL) and 30 μL SDS (10%). Resuspend gently.
7. Incubate 2 hours 30 minutes at 37°C.
8. Add 100 μL NaCl 5 M and 80 μL of the mix NaCl-CTAB (NaCl 0.7 M and CTAB 10%). Preheat the mix to solve it.
9. Incubate 10 minutes at 65°C.
10. Add 600 μL phenol.
11. Gently shake manually for 10 minutes.
12. Centrifuge 10 minutes at 10,700 rpm.
13. Transfer the upper aqueous phase to a new tube and add an equal volume of chloroform-alcohol isoamyl (24:1).Gently mix by vortex.
14. Centrifuge 5 minutes at 10,700 rpm.
15. Transfer the upper aqueous phase to a new tube.
16. Add 0.6 V of isopropyl alcohol and incubate at least 15 minutes at -80°C or overnight at -20°C.
17. Centrifuge 20 minutes at 10,700 rpm. Remove supernatant.
18. Wash the pellet with 1 mL ethanol 70% (2 or 3 times).
19. Centrifuge 15 minutes at 10,700 rpm. Remove supernatant with pipette.
20. Dry the pellet under vacuum for 20 minutes.
21. Resuspend the pellet on 30 μL TE or H₂O.
22. Add 2 μL RNAse and incubate 2 hours 30 minutes at 37°C.
23. Store the tubes at -20°C.

Miniprep

We are using the [http://www.sigmaaldrich.com/life-science/molecular-biology/dna-and-rna-purification/plasmid-miniprep-kit.html GenElute™ Plasmid Miniprep Kit] of Sigma-Aldrich according to the manufacturer directions.200px|thumb|center

Electroporation

Electro-competent cells of E. coli DH5α are electroporated at 1.25 mV, immediately we add SOC medium to the bacteria and we incubated on a shaker at 37°C for one hour, then, we culture cells in a solid medium containing the antibiotic of selection.

Primer Design

Primers were designed manually using [http://www.idtdna.com/analyzer/applications/oligoanalyzer/ OligoAnalyzer] of IDT, secondary structures were also calculated using [http://mfold.rna.albany.edu/?q=mfold/ mfold] and annealing conditions usign [http://primerdigital.com/fastpcr.html FAST PCR].

PCR - Pfu DNA polimerase

One reaction.

PCR - Taq DNA polimerase

One reaction.

If needed add 1 μL of DMSO per reaction, and adjust the amount of H₂O to a final volume of 10 μL.

PCR - Colony

Based on the protocol used with Taq polimerase, add 1 μL more of H₂O and take a few cells from the strain pricking it with a toothpick and mixing them with the other PCR reactives instead of using purified DNA.

PCR - Boiling

We make a lysate of the cells and add 1 μL of that lysate instead of purified DNA using the Taq polimerase protocol. To obtain the lysate, scrape the strain and resuspend the cells taken on 50 μL of H₂O, then, put at 95°C for 10 minutes.