mideterm 2 review

mideterm 2 review - Answer for Midterm 2 review questions:...

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Answer for Midterm 2 review questions: What size is the target insert vector? 2+6+3=11kb What size is your target insert? 2kb Your boss want Your Favorite Gene to be inserted into a vector that will allow both selection and screening of e.coli colonies transformed with the correct plasmid. You rifled through the freezer and found Target Vector. Is this vector what you’re looking for? Why or why not? Yes because it allows selection of colonies that have an ampR vector and screening for vector with an insert because the B-gal gene will be inactivated You decide to stick your target insert (YFG) into the target vector. Using what you know about restriction enzymes, gel electrophoresis, and ligation, how would you do this? You would restriction digest both vectors with HindIII +BamHI (Eco won’t work because it cleaves the target vector 2x) and run the digests on a gel. Cut out the band that is 2kb from target insert and ligate to the linear 12kb band from the target vector. Transform this into e.coli and select +screen How will you select for colonies that carry the target vector? Grow them on ampicillin coated plates-if the bacteria have the vector they will be able to grow and you will see a colony How will you screen for colonies that carry the correct vector AND the correct insert? Is there anything that needs to be special about the e.coli they are transformed into? Also plate onto X-gal plate-- colonies that are growing and white means the B-gal gene got interrupted by insertion of “your favorite gene (YFG).” If the colonies are blue it means that they have the vector (are growing on the ampicillin) but they do not contain your insert most likely because the vector relegated to itself not to your gene. Key-X-gal to be cleaved not only do you have to have the B-gal fragment in your vector, but the bacteria you transform your vector into has to have the other fragment so that B-gal can be functional and cleave x-gal. What size is the resulting vector + insert (YFG)? 14kb You want to double check that your insert has made it into the target vector in the correct orientation so you decide to do PCR and send the resulting DNA away for sequencing. Draw where you would design your primers to anneal to. You want your primers to be within the vector so when you get the sequence either side of your sequence will be sequence from the vector that you already
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know. For example lets say your vector sequence is AAATTTT right before where your insert starts and your insert starts with AGGGG and ends with TTTGAT. If your insert was put in the correct orientation your sequence (if using primer in vector) should be AAATTTTAGGGG. If your insert was flipped and not in the correct orientation then the sequence would read AAATTTTTAGTTT, the last couple nucleotides of the insert being reversed because the insert was put in the
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This note was uploaded on 07/08/2009 for the course LIFESCI LS 3 taught by Professor Paulolague during the Fall '09 term at UCLA.

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mideterm 2 review - Answer for Midterm 2 review questions:...

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