Question

QUESTION 2 1. In the Qiagen DNA purification system that we used with EcoRi-cut Yeast genomic DNA in the last Lab A. Why is it necessary to "purify" the EcoRl-cut Yeast DNA before proceeding to the DNA "cloning" steps we are about to do today? (1 pt) B. Which major type(s) of macromolecule within the restriction digest/binding buffer (PB) mixture bind(s) to the resin in the Qiagen "spin column," and which type(s) flow(s) through? (0.5 ptea)

Answer 1

A. It is necesary to remove the restriction enzyme once digestion of DNA is completed. This is because the restriction enzymes do have affinity for DNA and would interfere in subsequent reactions, in this case, cloning of Eco R1 digested yeast DNA into a vector.

B. Spin columns in most DNA purification kits usually are made up of silica that bind DNA. So when we purify restriction digests through the spin column, the DNA binds to the column whereas the restriction enzyme (protein) does not bind and is collected in the flow-through. The DNA boud to the column is then eluted using nuclease free water or TE (low-salt) buffer after a wash step.