MCB 120L Exp 7 Key (Q’s 1-3) 1. Restriction enzyme digest and agarose gel analysis a) Was an insert observed for the Pst I/Xba I double digest? Yes , in the (+) culture: pASK75/BVR b) If yes, then what approximate size, in bp, was observed? Approx 1000 bp was observed. c) Is this approximately the expected size, in bp? (What is the size of the cyanobacterial slr1784 gene?) The protein is 328 aa x 3 bp/aa codon = 984 bp. 984 bp + 30 added nucleotides on the 5’ side of each primer outside the coding region = 1014 bp Thus, the size of the observed band is what was expected. 2. SDS-PAGE and Western blot analysis a) Was a protein band observed in the presence of the BVR insert? Yes, a unique band was observed in the + lane (the negative control, - lane, and the + lane showed a similar band of lower molecular weight, thus was eliminated as a possibility) b) If yes, then what approximate molecular weight was observed? The band was between the 34 kD and the 43 kD MW markers; thus approx 38 kD.
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