Restriction Enzyme analysis of pUC19

Why are plasmids easier to isolate in their pure form than host chromosomes?

They are smaller and in greater number

Enzymes bind to what kind of DNA sequences?

Palindromic DNA sequences

• Sequences that read the same backward as forward, ex. kayak

How do most restriction enzymes function as?

Homodimers (one cleaving the top strand and one cleaving the bottom strand)

What does the restriction enzymes cleave?

the phosphodiester backbone

What are the enzymes used this experiment?

Pvull (creates blunt ends)

Bgll (creates sticky ends)

What is the plasmid used in this experiment?

pUC19

What are the components of the restriction enzyme digest?

Buffer (10X to 1X, 2 uL in each), DNA (ex. 65.2 ng/ul to 25 ng/ul), Enzyme (10,000 units per ml, so 10 units per ul, so 1 uL of each enzyme per reaction, so if you have both enyzmes, one enzyme each), the rest is nuclease free water

What are the four reactions total?

1. Uncut pUC19

2. Cut by Pvull (single digest pUC19)

3. Cut by Bgll (single digest pUC19)

4. pUC19 double digested with both Pvull and Bgll

What’s the charge of DNA?

negative

What is agarose gel electrophoresis used for?

For seperation of DNA, RNA, and proteins based on size and electrical charge

What’s bigger, DNA or protein?

DNA

What is meant by the topology of a plasmid?

physical shape and three-dimensional configuration

• specifically how double-stranded circular DNA is twisted, coiled or broken

When is a plasmid longer than the typical plasmid? When is it shorter?

Longer —> when its nicked

Shorter —> when its supercoiled