2. You have received a plasmid vector (Vector A) from a friend that contains You

Question

2. You have received a plasmid vector (Vector A) from a friend that contains Your Favorite Gene cloned into the EcoR1 and BamHl sites in the polylinker, as shown below. However,it is a LEU2 marked plasmid, and you want to transform it into a LEU2+ strain of yeast. So you can't use the LEU2 marker to select for cells that have received the plasmid. However, your yeast happens to be ura3- so you decide to move your favorite gene to a similar vector (vector B), except it has the URA3 marker instead Which enzyme(s) could you use to most casily cut out YFG from vector A, and open vector B in order to ligate YFG into vector B? Note, most of the enzymes that you think at first might work won't, for various reasons. Some of the other combinations might work, but they are not the easiest way to do it. You can assume that since the restriction sites in the polylinker were engineered to be close together, that the length of the polylinker sequence is negligible (it is probably about 50 base pairs, really.) The bases in vector A are numbered clockwise as shown (starts with 0 and ends at the same place at 3000). Hint 1: consider all the pieces you will get when you cut with your chosen enzymes. How easy will it be to identify the fragment you want? Hint 2: consider which buffer to use to make a digestion using 2 enzymes work best. Assume that any enzyme used with a nonoptimal buffer will cut poorly. Hint 3:Whatever enzymes you cut the piece out with, the piece will have those sticky ends, so you want to use the SAME enzymes to cut open the recipient vector see plasmid maps on next restriction enzyme cuts best in buffer HindIII Xbal For each enzyme that you rejected using, give each reason why it won't work. (Some enzymes have one reason, and some have more than one reason.) Before you ask anyone for help.give it some thought If you

Explanation / Answer

If we look at the vector map of the LEU2 vector we will find that we cannot use SacI, SalI, PstI and HindIII as there are two sites of the mentioned enzyme in the vector. So we need to cut the gene with EcoRI, XhoI or XbaI. But upon looking at the URA3 vector we will find that it has two sites for XbaI. So we cannot use XbaI for cloning.

We can use two strategies to clone our desired gene into the URA3 vector.

1- Cut the gene of interest from the LEU2 vector by EcoRI and at the same time cut the URA3 vector also with EcoRI enzyme.

For checking the directionality of the clone we need to use Colony PCR strategy- In this, we will use the reverse primer that is designed from the insert and Forward primer from the vector. If we get the PCR product of the desired size then our clone is confirmed otherwise not.

2- Alternatively we need to digest the insert and URA vector sequence with EcoRI and XhoI as the buffer of both the enzymes are not compatible with each other and ligate the digested insert and URA3 vector

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