Biochemistry Lab Exercise | My Assignment Tutor

Biochemistry Lab Exercise 2, page 1/5 Student ID number:______________ Lab day/time: ______________ Overall mark: ( /20) Restriction Enzyme Exercise: Given the following information: Uncut lambda () DNA is 48,502 base pairs long.DNA is normally in a linear state before entering a host cell. However, on infection of a host cell theDNA circularises. To be able to circularise, the lambda DNA has sticky or cohesive (cos) sites on each of it ends to allow it to anneal together. When heated (i.e. in a test tube), the cohesive ends will split apart, causing the  DNA to become linear again. The point at which the  DNA splits and becomes linear again is classed as 0 base pairs and is where you begin counting from.HindIII cutting sites on  DNA occur at: 23,130; 25,157; 27,479; 36,895; 37,459; 37,584 and 44,141 base pairs.EcoRI cutting sites on DNA occur at: 21,226; 26,104; 31,747; 39,168 and 44,972 base pairs.You can cut using a mixture ofHindIII and EcoRI, or each enzyme individually. Determine the number of DNA fragments (bands), and their expected sizes, you would observe for each restriction enzyme when cutting  DNA and enter them in the following table. Show calculations here: Single cut HindIIINumber of separate DNA fragments/bands:Band sizes:Single cut EcoRINumber of separate DNA fragments/bands:Band sizes:Double cut HindIII and EcoRINumber of separate DNA fragments/bands:Band sizes: These are the fragments/bands that you would expect to see if you ran an agarose gel. Marks: ( /4) Biochemistry Lab Exercise 2, page 2/5 Student ID number:_______________ Agarose Gel Labelling Exercise Below is a gel image similar to the one that would be produced in this experiment. On the image, label the size of as many DNA fragments as possible using the information obtained from the molecular marker and your calculations in table 1 (page 1). Marks: ( /2) Biochemistry Lab Exercise 2, page 3/5 Student ID number:_______________ Questions: What charge does DNA carry? Why? Marks: ( /2) Define gel electrophoresis and the mechanism behind DNA fragment separation. Marks: ( /2) List how you could alter DNA fragment separation? Marks: ( /2) Why can’t you see the 3-dye marker on the gel photo? What is the purpose of the 3-dye marker in agarose gel electrophoresis? Marks: ( /2) What is the purpose of the molecular weight (MW) marker in agarose gel electrophoresis? Marks: ( /2) Biochemistry Lab Exercise 2, page 4/5 Student ID number:_______________ Spectrophotometric Measurement of DNA Absorbance of Lambda DNA Uncut lambda DNA 260 nm: 280 nm:Cut with HindIII 260 nm: 280 nm:Cut with EcoR1 260 nm: 280 nm:Cut with EcoR1 and HindIII 260 nm: 280 nm: What was the concentration for each of your DNA samples? Show your calculations. Marks: ( /2) Was each of the DNA preparations pure? Would you use these samples for further DNA analysis? Marks: ( /2) Biochemistry Lab Exercise 2, page 5/5 Student ID number:_______________ This page has been left intentionally blank. You may use this page for any notes or calculations relating to the lab.

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