Question

How did you EXPECT your uncut lane to look in the gel image? What ACTUALLY happened? What is a plausible explanation if there was any discrepancy?

How did the observed BclI and EcoRI digest results compare to the expected results? If they differed, list a potential reason why?

BclI was incubated at 50°C, while EcoRI was incubated at 37°C. Why was this necessary? What might you predict to occur if you accidentally switched the temperatures?

3) Using the DNA ladder, estimate the experimental size of all bands observed in the sample lanes. (3pt) Gel Lane Plasmid Number of bands observed Estimated size (bp) of each band 2 (tube 1) A 3 (tube 2) B 4 (tube 3) A 5 (tube 4) B 6 (tube 5) A 7 (tube 6) B Restriction Enzyme N/A, uncut N/A, uncut Bcll Bell ECORI ECORI

Answer 1

The optimal temperature of BcII activity is 50oC. However, it is half active at 37oC. The optimal temperature of EcoRI activity is 50oC. However, it is half active at 37oC. The optimal temperature of EcoRI activity is 37oC. If the temperature is different , the activity of EcorI decreases.  So, it is necessary to incubate BcII  at 50oC and EcoRI at 37oC. If the temperature is switched accidentally, activity will decrease.