The following restriction digestions were set up: In tube 1: 3 µL PCR, 6 µL wate
ID: 3164725 • Letter: T
Question
The following restriction digestions were set up: In tube 1: 3 µL PCR, 6 µL water, and 1 µL 10X buffer, In tube 2: 3 µL PCR, 5 µL water, 1 µL 10x buffer, and 1 µL Sac I enzyme. The two tubes were incubated in a 37°C water bath for 15 minutes. The reaction was halted by adding 2 µL of 5x loading buffer and mixed by tapping the tube. The tubes were briefly centrifuged to move all of the liquid to the bottom of the tube.
Lane 1 is the ladder. Lane 2 contains tube 1. Lane 3 contains tube 2. (Ignore the other lanes)
What do the bands represent in lanes 1 and 2? Did the PCR product give a single band? Was the negative control lacking a band? Was the plasmid cut by the Sac I enzyme? Do the Sac I fragments add up to the size of the PCR product?
Explanation / Answer
Bands in lane 1 represent different DNA fragments of known sizes. Single band in lane 2 (lesser migration) in the upper region than all the bands in lane 1 suggests that the size of the PCR product is greater than the highest size band in lane 1.
The PCR product gave a single band. Negative control has a single band, suggesting there is no restriction.
Sac I enzyme has cut the DNA. Lane 3 produced two bands due to the restriction.
Since the sizes of the bands in ladder are not given, I cannot be sure whether they add up to the size of band in lane 2. However, from the gel picture, it looks like the sizes of bands in lane 3 would add up to size of band in lane 2.