Consider the following information to answer questions 14 Yo

Consider the following information to answer questions 1-4:

You successfully amplify the psy2 sequence and wish to clone the PCR fragment into vector 2 in order to express it in yeast. The cloning sites available on this vector are shown below.

There are two different ways to insert the amplified psy2 sequence into vector

1. Give the restriction enzyme(s) that you could use to cut the vector and the psy2 coding sequence for each of these.

2. Directional cloning: Cut vector and psy2 with:

3. Non directional cloning: Cut vector and psy2 with:

4. Which enzyme (s) could you use to verify the presence and orientation of the insert?

Answer all of these, help explain

Stu Promoter Sall EcoRI Start EcoRI 5\' GTC GACGGAATT CAG GCCT CGT CGA CTC CGGC 3\' Vector 2 Psy2 3 CA GCT GCC TTAA GTC CGGA GCA GCT GAGGCCG5\' Stul: 5\'- AGG/CCT-3\' Sall: 5\'-G/TCGAC-3\' EcoRI: 5\'-GAATTC-3\' BamHI: G GATCC

Solution

Answer:

1. The vector and insert can be cut with StuI and SalI in combination or with SalI alone. But in the second case with only SalI, there is 50% probability that the sequence get inserted in the correct orientation. EcoR1 cannot be used since the insert has the EcoR1 site within the Psy2 gene.

2. For directional cloning, cut vector with: StuI and SalI

cut insert with: StuI and SalI

3. For non directional cloning, cut vector with: SalI

cut insert with: SalI

Non-directional cloning can be performed with a single restriction enzyme. In this case, SalI is suitable since the insert contains two different SalI sites on either side of the insert.

4. StuI and SalI can be used to verify the presence and orientaion of the insert. This is because its directional with the reading frame of the insert in the vector.