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. 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 ± State lab Goal(s) . ± Outline or make a flow chart of the methods. ± Complete all calculations. Colony with pET28b-EGFP
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87 During this lab you will analyze the size of your amplicons from lab C1 through agarose gel electrophoresis and select a clone with the appropriate size amplicon. You will inoculate a culture of the selected colony from your master plate so that you can isolate plasmid and perform a restriction analysis of your selected clone in Lab C3 and C4. Restriction Analysis is normally the next step in the determination of the authenticity of a clone. Materials Horizontal electrophoresis tank with casting tray, 10 teeth comb, and lid Agarose 1.2% agarose gel in 1X TAE 10X TAE buffer λ BstEII 1 Kb plus DNA standard Amplicons from lab C1 SYBR green 6X Tracking DYE Master plate from C1 One Culture tube with LB broth + KAN30 Procedure a.
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2010 Bio 311 Lab C Manual (part 2) - C2. Analysis of PCR...

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