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Team:Colombia/Notebook/Protocols
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==='''PCR - Pfu DNA polimerase'''=== | ==='''PCR - Pfu DNA polimerase'''=== | ||
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One reaction. | One reaction. | ||
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One reaction. | One reaction. | ||
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==='''PCR - Colony'''=== | ==='''PCR - Colony'''=== | ||
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Based on the protocol used with Taq polimerase, add 1μL 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. | Based on the protocol used with Taq polimerase, add 1μL 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. | ||
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| + | ==='''PCR - Boiling'''=== | ||
Revision as of 22:26, 13 July 2012
Contents |
Protocols
Bacterial DNA extraction protocol
Miniprep
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
PCR - Pfu DNA polimerase
One reaction.
| Reactives | Amount (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
One reaction.
| Reactives | Amount (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 10μL.
PCR - Colony
Based on the protocol used with Taq polimerase, add 1μL 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.
