1. PCR (Polymerase Chain Reaction): PCR used to modify Lambda_cI by copying a section of the gene, adding RBS, and using RS (restriction site enzyme) to cut copied gene section at the appropriate restriction site.
PCR Components in 50uL sln
| Components | Volume
|
| Phusion HF Buffer | 10.0uL
|
| 10 mM dNTP's | 1.0uL
|
| Primer mix | 2.5uL
|
| Template DNA | 1.0uL
|
| Phusion DNA Polymerase | 0.5uL
|
| H20 | 35.0uL
|
| 2. Run PCR in Gel Electrophoresis:
|
| a. Used 0.8% agarose gel, TAE buffer, and ethidium bromide (interculating agent)
|
| b. Gel proved have correct Lambda_cI gene with 750 base pairs. Can now perform PCR purification (add buffers).
|
| 3. Miniprep MCherry gene
|
| 4. Spectrophotometry: Used to determine the concentration of DNA in solution. Spec'd Lambda_cI with RBS (PCR sample).
|
|
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