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Team:Colombia/Notebook/Protocols
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==='''PCR - Boiling'''=== | ==='''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 | + | 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 growth solid culture and resuspend the cells on 50 μL of H<sub>2</sub>O, then, put at 95°C for 10 minutes. |
Revision as of 22:26, 14 July 2012
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Contents |
Protocols
Bacterial (Xam) DNA extraction protocol
- Shaking culture overnight.
- 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.
- Wash the pellet twice with 1 mL NaCl 1 M and Resuspend.
- Centrifuge 2 minutes at 10,700 rpm and remove the supernatant.
- Resuspend the pellet on 567 μL TE (10 mM Tris-HCl pH 8; 1 mM EDTA pH 8).
- Add 3 μL proteinase K (20 mg/mL) and 30 μL SDS (10%). Resuspend gently.
- Incubate 2 hours 30 minutes at 37°C.
- 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.
- Incubate 10 minutes at 65°C.
- Add 600 μL phenol.
- Gently shake manually for 10 minutes.
- Centrifuge 10 minutes at 10,700 rpm.
- Transfer the upper aqueous phase to a new tube and add an equal volume of chloroform-alcohol isoamyl (24:1).Gently mix by vortex.
- Centrifuge 5 minutes at 10,700 rpm.
- Transfer the upper aqueous phase to a new tube.
- Add 0.6 V of isopropyl alcohol and incubate at least 15 minutes at -80°C or overnight at -20°C.
- Centrifuge 20 minutes at 10,700 rpm. Remove supernatant.
- Wash the pellet with 1 mL ethanol 70% (2 or 3 times).
- Centrifuge 15 minutes at 10,700 rpm. Remove supernatant with pipette.
- Dry the pellet under vacuum for 20 minutes.
- Resuspend the pellet on 30 μL TE or H2O.
- Add 2 μL RNAse and incubate 2 hours 30 minutes at 37°C.
- 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
We electroporate E. coli DH5α Electro-competent cells at 1.25 mV, then we resuspend the cells in SOC medium and incubate on a shaker at 37°C for one hour, then, we plate the cells in a solid medium containing antibiotic 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.
| Reactives | Volume(μL) |
|---|---|
| H2O | 17.95 |
| Buffer | 2 |
| MgSO4 | 1.6 |
| dNTP's | 0.4 |
| Primer Fw | 0.4 |
| Primer Rv | 0.4 |
| Taq | 0.06 |
| Pfu | 0.19 |
| DNA | 2 |
| Total | 25 |
PCR - Taq DNA polimerase Invitrogen
One reaction.
| Reactives | Volume(μL) |
|---|---|
| H2O | 6.15 |
| Buffer | 1 |
| MgCl2 | 0.5 |
| dNTP's | 0.25 |
| Primer Fw | 0.5 |
| Primer Rv | 0.5 |
| Taq | 0.1 |
| DNA | 1 |
| Total | 10 |
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 growth solid culture and resuspend the cells on 50 μL of H2O, then, put at 95°C for 10 minutes.
