Chapters 18 and 19 Problems

Chapters 18 and 19 Problems - Name: Hellums, Jessica UTEID:...

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Name: Hellums, Jessica UTEID: JSH575 Problems and Questions for Chapter 18 and 19 Material A2: a. Automated sequencing a. Must have ddNTPs, dNTPs, primer of single stranded DNA made from a DNA synthesizer or ordered as an oligo, DNA polymerase, and a template. The primer is complimentary to the plasmid right before the insert on the plasmid so that the insert is used as a template. At random, either a ddNTP or a dNTP will add and then after a ddNTP is added the strand is terminated. The ddNTPs fluoresce with a different color for each base and they lack a hydroxyl on the 3’ carbon so they cannot be further elongated once one is added. The fragments are of every possible length because it happens so many times and its run through a machine with a column where the fragments can move through and separate with the shortest fragment falling first and the longest last. The fragments drop in a solution where it is hit with a laser that causes the color to show of the respective ending ddNTP. Then the color is detected and the sequence can be determined by the successive colors observed. Then you do the sequenceing in the other strand with a primer complimentary to that one and check for the overlap of the gene you want to be sure you got the gene accurately. Accuracy declines as the process goes on so doing both strands promotes accuracy. Then compare the sequence to the NCBI Database and check to see if it matches another gene for that species or other species or genes of a family. b. Blue-white screening a. After bacteria have been transformed to take up the plasmid with the gene of interest, most of the bacteria do not actually transform. Ampicillin will remove bacteria that do not have the plasmid because the plasmid has an amp R gene. Then using X-gal and IPTG you can determine if the bacteria has a nonrecombinant plasmid or a recombinant
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This note was uploaded on 04/07/2008 for the course BIO 325 taught by Professor Saxena during the Spring '08 term at University of Texas at Austin.

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Chapters 18 and 19 Problems - Name: Hellums, Jessica UTEID:...

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