Question

Suppose you are going to do a restriction digest with a plasmid, using the restriction enzyme Eco R1. A map of the plasmid is shown here. The entire plasmid is 6000 bp, and there are Eco R1 restriction sites at 1500 bp, 2000 bp, and 4000 bp. You’re going to run the entire volume of the digest on a gel, and you want to cut just enough DNA to have 50 ng in the smallest band on your gel.

Starting DNA concentration: 50.0 μg/ml

Eco RI comes as a stock solution at 10,000 Units/ml, and 1 Unit will cut 1 μg of DNA. The enzyme comes with a separate tube of 10x enzyme buffer.

Give the volumes of everything you will put in the restriction digest tube. The final volume of your restriction digest should be 20 μl. Use realistic numbers, as if you are actually doing the experiment. Use an appropriate number of significant figures.

DNA volume: μl

Eco RI enzyme: μl

Buffer: μl

Water: μl

Answer 1

EcoRI expect to see three bands: the linearised vector (6kb), the 5' end of the insert (0.5 kb) and the 3' end of the insert (3.5 kb). In order to see the smallest band (0.5 kb) you want it to contain at least 50 ng of DNA. The smallest band is 1/20th the size of the uncut plasmid. Therefore you need to cut 20x50 ng, that is 1000 ng of DNA (1µg). Then your three bands will contain 600 ng, 50 ng and 350 ng of DNA respectively. All three bands will be clearly visible on the gel.

As we have 50$\mu$g/ml so, we will need 5$\mu$l of stock DNA. We need 1$\mu$l of EcoR1 for 1$\mu$g of DNA. Buffer will be 3$\mu$l of 10X buffer. And rest 11$\mu$l will be covered by water.