1. The final elution of your miniprep plasmid DNA has a concentration of 250 ng/uL. You need to load 500 ng on an agarose gel to be able to see the DNA. You plan to assemble a single restriction digest reaction (cutting the plasmid with EcoRI only) with a total volume of 20 uL, and incubate it in a 37oC waterbath for 5 min. What is the minimum amount (in uL) of FastDigest EcoRI enzyme you need to use?

What is the volume of plasmid DNA, in uL, that you would use in the reaction?

2. For Lab 5 he 203L preproom needed to restriction digest a large quantity of lambda DNA in order to have enough for all the students. The total amount of DNA required for one reaction was 22 ug. Starting with the basic protocol, scale up the reaction for the larger amount of DNA. How mouch enzyme (in uL) should be used?

What is the recommended total reaction volume?

3. An SDSU grad student requested a plasmid fom a research lab at UCSF. It expresses a protein of interest from a human gene approximately 242 amino accids in length that has been cloned into a plasmid expression vector called pET15b, sold by Novagen. She decides to do a restriction digest on the plasmid to verify it contains the gene by cutting th vector on each side of where the gene was inserted. The UCSF alb told her the restriction enzymes they used for cloning were Hind III and Ndel. The SDSU lab has the Hind III enzyme in the freezer and doesn't have Ndel. The grad student decides that as long as she cuts it with Hind III and knows the whole size of the plasmid, she can still get data the same day to know if the gene is there. She goes to the internet and downloads a vector map of pET15b to find its size. When she does the digest and runs it on an agarose gel, what size band (bp) should she expect to see if the plasmid has the gene?