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2010 Bio 311 Lab C Manual (part 2)

2010 Bio 311 Lab C Manual (part 2) - C2 Analysis of PCR...

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86 C2. Analysis of PCR Products Lab Overview: Last time you resuspend colonies from your experimental plate in water and used the heated crude plasmid DNA for polymerase chain. You also created a master plate of the selected bacterial clones for future use. For this lab, you will view your PCR products (amplicons) on an agarose gel during lab C1. You will also select the SCI that has the pET28b-EGFP from your master plate and grow it up for further analysis next week. Background In Lab B, you were to create the pET28b-EGFP construct through restriction digestion and ligation procedures. You transformed a transgenic strain of E. coli , which is designated DE3, with your ligation mixture or with the actual plasmid pET28b-EGFP that you were trying to create yourselves. DE3 strains express the T7 RNA polymerase in the presence of inducers of the lac operon such as IPTG. The T7 RNA polymerase will transcribe a gene inserted correctly in the pET28b vector because the vector has transcription promoter, initiation and termination sequences that are recognized by the T7 RNA polymerase (review the plasmid map of pET28b) in the Appendix. In the pET28b-EGFP construct, the insert gene is the one for EGFP and so you looked for a colony that fluoresced green under blacklight. In Lab C1, You selected colonies for analysis and performed the technique called polymerase chain reaction (PCR). These selections should have been based on the following criteria: 1. The transformed bacteria acquired resistance to the antibiotic Kanamycin. 2. The transformed DE3 bacteria produced the EGFP in the presence of IPTG. Enhanced Green Fluorescent protein Kanamycin resistance protein Bacterial host proteins T7 RNA Polymerase Read "An Overview of the BCE labs" and Lab C2. State lab Goal(s) . Outline or make a flow chart of the methods. Complete all calculations. Colony with pET28b-EGFP
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