Build-a-Gene Session 4: Difference between revisions
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== LIGATION == | == '''LIGATION''' == | ||
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3. Incubate at 80C for 20 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. | |||
Revision as of 07:43, 12 August 2013
LIGATION
This step will allow us to join the vector, the promoter, and the emGFP gene together into one circular DNA molecule.
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.
