4. You are attempting to clone an EcoRI fragment into an EcoRI-restricted vector

Question

4. You are attempting to clone an EcoRI fragment into an EcoRI-restricted vector treated with CIP, and you obtain the following results on your transformation plates (with Amp):

#1    vector only +ligase                   107 colonies

#2 vector+insert+ligase                   663 colonies

1) would you expect more, fewer or about the same number of colonies if no ligase were added in #2? Explain.

2) would you expect more, fewer or about the same number of colonies with #1 with a vector not treated with CIP ? Explain.

Explanation / Answer

Treatment of the EcoRI-restricted vector with CIP allows the cloning of EcoRI fragment by inserting its copy into it generating a transconstruct and by its multiplication into suitable host. The completion of the transconstruct is done by sealing the open end created by the CIP(restriction enzyme) by adding up ligase enzyme which carries out the sealing activity. Now if we consider the first case (1) wherein no ligase was added in #2 the number of colonies expected would vary in different sets of results as ligase seals the open end justifying that the desired gene to be cloned is within the vector. Thus no ligase would guarantee up the presence or successfull transfer of desired gene in the vector. Thus it can be stated that by not adding ligase the expected number of colonies would be fewer. Proceeding with second case (2) wherein no CIP action was done in #1 the number of colonies expected would decrease as the entry of desired EcoRI fragment would be prohibited as the vector was EcoRI restricted.

Related Questions

Navigate

• Browse (All)
• Browse 4
• Subjects

Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.