reading an agarose gel FIGURE 14.3 Restriction digest experiment. CHayden-McNeil
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Question
reading an agarose gel
FIGURE 14.3 Restriction digest experiment. CHayden-McNeil, LLC Answer the questions using the information below. 1. 2. 3. What is in the upper band in lane D? What is in the lower band in lane D? What is in lanes B, C, and F? Can you estimate how many bp are in these bands from the DNA ladder? 4. What is in lane E? How many bp are in each of these two bands? Which would be the "vector" band and which would be the "insert" band? 5. (Graduate-level question) If the vector and insert bands contain the same number of DNA fragments (and they must), why is the insert band fainter? Definitions Super-coiled: Circular, super-coiled DNA migrates more rapidly than linear DNA of the same size. Plasmid DNA is normally super-coiled Nicked, circular: One of the two strands of the double-stranded DNA is broken. DNA uncoils, but remains circular (open circular) and usually migrates more slowly than linear DNA of the same size. Digest with one enzyme: One piece of linear DNA Digest with two enzymes: Two pieces of linear DNA; one is the size of the gene inserted between the two enzyme sites in the multi-cloning site (insert band), and the other is the size of the rest of the plasmid (vector band).Explanation / Answer
a. Upper band is nicked coiled as it migrates the slowest.
b. The lower band in D represents supercoiled that runs the fastest.
c. B,C, F lanes have a single restriction enzyme digested DNA which gives a single band. If the intense band (3rd from the bottom) corresponds to 500 bp, then the digested product is at 3.5 kb
d. lane E is a double digested product correponding to size of vector as 2.5 kb and the release or other digested product is 100 bp approx. Top would be the vector band and bottom insert band.
e. Insert band is generally faint due to incomplete digestion . Else if the size of both vector and insert is exactly the same, they generally give same level of EtBr UV fluorescence. vector generally takes in more of interacalated dye compared to insert.
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