DNA Structure
Today:
1) Set up PCR (p. 164) – do first
- Quiz on this lab (RPP, PCR) will be Wednesday after Spring Break.
2) Recombinant paper plasmids (p. 189)
For Monday:
- Turn in page 190 (maps, showing ALL restriction sites) and tell what enzyme you chose.
- Turn in completed recombinant plasmid (with chromosomal DNA inserted).
- For each part of the lab, understand what we did (methods) and why.
Background:
Bacteria have a single, circular chromosome.
Some bacteria also have plasmids: a small circular piece of DNA carrying only a few genes.
- They aren't essential for survival, but they often help.
- Ex. some give the bacteria resistance to antibiotics.
As we saw before with the bacteriophages, some viruses can inject their genes into bacteria,
turning them into virus-producing factories.
Over time, bacteria have developed weapons against such DNA invasions. These are called
restriction enzymes: enzymes that recognize and cut up foreign DNA.
- They always cut at specific (& symmetric) base sequences.
- Discovered in the late 1960s: a big advance (now > 3000 have been identified).
- Researchers have learned to use these enzymes to produce DNA fragments.
- These can be used to make a DNA “fingerprint.”
- They can also be used to make recombinant DNA.
Part 1: PCR (See lab manual page 163)
Part 2: Recombinant Paper Plasmids (page 189):
Our goal is to cut out a piece of chromosomal DNA that codes for the production of a protein
and then insert it into a bacterial plasmid.
Cut plasmid DNA strips (p. 191) and tape them together in any order, then make a circle.
Cut chromosomal DNA strips (p. 193) and tape together in the order in which they are given so
that the protein gene is intact (rows 2-4).
There are several possible restriction enzymes to choose from (p. 195) and you need to pick the
most appropriate one for your particular plasmid and chromosomal DNA.
- Locate all the restriction sites on both of your DNA pieces and mark them on the map
provided (page 190). Remember they cut at ALL available restriction sites.
- Determine which enzyme to use. We must not cut the origin of replication or the
protein-coding region, and we want to cut one of the antibiotic resistance sites.
- Cut at all the sites and make your recombinant plasmid. Turn this in on Monday.