Build-a-Gene Session 4: Difference between revisions
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== LIGATION == | == LIGATION == | ||
The restriction enzyme digest that we completed last week generates DNA molecules with single stranded regions, termed "sticky" ends. When these molecules are combined, complementary bases can hydrogen bond, bringing the DNA molecules together. The enzyme DNA ligase will then create a phosphodiester bond, joining the backbone of one DNA molecule to the backbone of the other DNA molecule in a covalent bond. This step will allow us to join the vector, the promoter, and the emGFP gene together into one circular DNA molecule. | The restriction enzyme digest that we completed last week generates DNA molecules with single stranded regions, termed "sticky" ends. When these molecules are combined, complementary bases can hydrogen bond, bringing the DNA molecules together. The enzyme DNA ligase will then create a phosphodiester bond, joining the backbone of one DNA molecule to the backbone of the other DNA molecule in a covalent bond. This step will allow us to join the vector, the promoter, and the emGFP gene together into one circular DNA molecule. For more information on restriction enzyme digests and DNA ligation: [http://www.dnalc.org/resources/animations/restriction.html] | ||
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This step will allow us to transfer the vector containing the emGFP gene and promoter into bacterial cells. This will separate the individual DNA molecules because almost all cells will pick up only one DNA molecule (we are therefore "cloning" the DNA). As the bacteria grow, they will continue to replicate the introduced DNA as they replicate their own bacterial chromosome. This will lead to amplification of the DNA as the cells grow. | This step will allow us to transfer the vector containing the emGFP gene and promoter into bacterial cells. This will separate the individual DNA molecules because almost all cells will pick up only one DNA molecule (we are therefore "cloning" the DNA). As the bacteria grow, they will continue to replicate the introduced DNA as they replicate their own bacterial chromosome. This will lead to amplification of the DNA as the cells grow. For more information on the mechanism of transformation: [http://www.dnalc.org/resources/animations/transformation2.html] | ||
Revision as of 07:50, 12 August 2013
LIGATION
The restriction enzyme digest that we completed last week generates DNA molecules with single stranded regions, termed "sticky" ends. When these molecules are combined, complementary bases can hydrogen bond, bringing the DNA molecules together. The enzyme DNA ligase will then create a phosphodiester bond, joining the backbone of one DNA molecule to the backbone of the other DNA molecule in a covalent bond. This step will allow us to join the vector, the promoter, and the emGFP gene together into one circular DNA molecule. For more information on restriction enzyme digests and DNA ligation: [1]
1. Combine all of the reagents below together into one thin-walled PCR tube.
| Vector | 1,5 ul |
| emGFP gene | 1.5 ul |
| Promoter | 1.5 ul |
| T4 Ligase + buffer mix | 5.5 ul |
| Total | 10 ul |
2. Incubate at 16C for 30 min
3. Incubate at 80C for 20 min
TRANSFORMATION
This step will allow us to transfer the vector containing the emGFP gene and promoter into bacterial cells. This will separate the individual DNA molecules because almost all cells will pick up only one DNA molecule (we are therefore "cloning" the DNA). As the bacteria grow, they will continue to replicate the introduced DNA as they replicate their own bacterial chromosome. This will lead to amplification of the DNA as the cells grow. For more information on the mechanism of transformation: [2]
