Practice problem . In question 3, I told you that I had cloned the Cox-1 gene of

Question

Practice problem . In question 3, I told you that I had cloned the Cox-1 gene of tardigrades into the TOPO cloning vector. I decided thatl wanted to do studies on the function of COX-1 so I had to subclone it into an expression vector. The Cox-1 gene only has a Hindlll restriction site. Alul, Dral, Fspl, andHpal form blunt ends. All others create overhangs. · A: (5 pts) Tell me what enzymes I need to use to cut each vector if the Cox-1 gene is in the TOPO vector in the correct orientation B: (5 pts) Tell me what enzymes I need to use to cut each vector if the Cox-1 gene is in the TOPO vector in the incorrect orientation TOPO plasmid Expression plasmid

Explanation / Answer

If Cox-I is in the correct orientation as shown in the given image (left side),

i. Digest the TOPO vector with Alu I or Dra I (both flank the insert) to elute the insert. Digest the expression plasmid with Fsp I and Hpa I (both generate blunt ends). Perform blunt end ligation.

ii. We can simply cut both vectors with Eco RI. This will create the problem of self-ligation.

If Cox-I is in the incorrect orientation,

i. Digest the TOPO vector with Bam HI and Xho I to release the insert. Digest the TOPO vector with Bam HI and Xho I. Perform sticky end ligation.

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