Question

We have been doing PCR to take DNA from Listeria monocytogenes and eventually add it to e coli. We had a vector of pET28a and our PCR insert was HupD. Prior to purifying the PCR insert, it had a bp of 1700. We then purified our vector using a Qiagen mini-prep kit. We purified our PCR insert using a Qiagen PCR clean-up kit. We put both DNA solutions in the freezer until next class. In the next class, we added restriction enzymes BamHI and XhoI to a solution of our DNAs along with buffer and sterile DI water. We incubated both solutions for an hour, then put them in the freezer until next class. Next class, we ran it through the agarose gel.

Question: In lab 8b, we need to run an agarose gel after the restriction digestion experiment. In the gel result, vector DNA is shown on the gel in a position closer to the top well than the one we did in Lab 7. Briefly explain the position change of vector DNA on the gel before and after the digestion experiment.

Answer 1

• After digesting with BamHI and XhoI, the band will be formed at 5.3 kb (slightly more migration than the undigested DNA) because the difference between the location of the recognition sites of BamHI and XhoI is 40 bp. So this 40 bp fragment will be removed from the plasmid.
• So 2 fragments will be formed after digestion
  1. 40 bp
  2. 5329 bp
  • Original size of plasmid is 5369 bp
• 40 bp band might not get resolved but the other band at 5329 will be appeared.