Question

help with questions 5 to 10 please

PCB 3023L Lab #4 Protocol & Worksheet (30pt) You may work in your lab groups durine class. but all written answers must be completed individually in your own words. 1) Using the plasmid map for orientation 1 and the cDNA map as a guide, complete the plasmid map for orientation #2. (4pt) 612 1318 1 - EcoRi EcoRI Xbal ECORV -Xbal- 1662 +Bell EcoRI EcoRV Not FP -- Xhol X 2015 PRSP + CDNA = 5541bp (orientation 2) Kanamycin Res

Answer 1

5) The expected and the observed results from both the endonucleases (Bcll and EcoRI)are the same indicating that the plasmid orientations were the same as observed from which the expected observations were taken.

6) The temperature used for incubation depends mainly on the thermal stability and the temperature dependence on the enzyme activity.As restriction endonucleases are proteins ,chances for denaturation are more when temperature fluctuation happens.There is an optimum temperature at which these endonucleases act (in which they show the maximum effeciency depending on their activation energy).This is 50 degrees for Bcll and 37 degree celcius for EcoRI.

7)Plasmid A is in orientation 1 and Plasmid 2 is in Orientation 2 comparing the band lengths derived from the first expected and compared results.So option "a" is the correct answer.

8) The length of the bands observed after digestion with EcoRI can be compared to confirm the result.

9) The digestions by Xhol may not be considered for determining the orientations because they presented only a single site for digestion and single fragment after digestion.

10)Xbal can be chosen to determine the plasmid orientation because they presented multiple (more than one ) restriction sites and therby number of fragments in the plasmid.