Experiment+14

Isolation of Recombinant Plasmid

-Transform DP1000 DNA into E.coli DH5 α (from StreptoC Pneu.) -Plate E.coli on TAXI plates -If growing and white then is recombinant with DNA inserted into pBluescript -pick some white colonies -sequence

TAXI plate - Thiamin,e Ampicilin, X-gal, IPTG

Pages 285-303

Forms of RNA (I think he said something about this in lecture.) Abundance of RNA types: Ribosomal > Messenger > Transfer

through pg 295 nothing worth knowing

1. Disruption of the cell wall and release of the DNA into a medium in which it is soluble and protected from degradation. 2. Dissociation of the protein DNA complexes 3. Separation of the DNA from other soluble cellular components.
 * Outline on DNA extraction:**

--Plasmid Replication modes Stringent replicated plasmids - present in only a few copies Relaxed replicated plasmids - present in many copies, sometimes up to 200 (in rare cases up to 3000! reaching 30~40% of cellular DNA)
 * Isolation of plasmid DNA:**
 * --**Plasmid size: 2x10^6 - 20^10^6 i.e. 3000 ~ 30,000 bp

- can be in covalently closed circular form - much smaller than genomic DNA
 * Plasmids have two main differences that are used in separation**

-specific interaction between plasmid DNA and a solid support (ex. nitrocellulose microfilters) - selective precipitation of chromosomal DNA,uses plasimd resistance compared with genomic DNA to temp, ph, etc.(denaturing agents). - sedimentation behavior (if highly purified)
 * Three categories of plasmid isolation**

Major absorbtion band 260nm protein 280nm A260/280 1.8 protein contamination < < upper bound?
 * DNA and UV**

intercalation - the process of ethidium bromide binding in between stacked base pairs.

Pages 309-326

Molecular Cloning

Recombinant DNA/Molecular Cloning - covalent insertion of DNA fragments from one type of organism into another organism


 * called cloning mostly to impress prospective high school students. ; ).

Restriction Endonucleases --Cohesive/sticky ends Overhangs and sticky ends Non-blunt ends are created by various overhangs. An overhang is a stretch of unpaired nucleotides in the end of a DNA molecule. These unpaired nucleotides can be in either strand, creating either 3' or 5' overhangs. These overhangs are in most cases palindromic.The simplest case of an overhang is a single nucleotide. This is most often adenosine and is created as a 3' overhang by some DNA polymerases. Most commonly this is used in cloning PCR products created by such an enzyme. The product is joined with a linear DNA molecule with 3' thymine overhangs. Since adenine and thymine form a base pair, this facilitates the joining of the two molecules by a ligase, yielding a circular molecule. Here is an example of an A-overhang: --blunt ends. The simplest DNA end of a double stranded molecule is called a blunt end. In a blunt-ended molecule both strands terminate in a base pair. Blunt ends are not always desired in biotechnology since when using a DNA ligase to join two molecules into one, the yield is significantly lower with blunt ends. When performing subcloning, it also has the disadvantage of potentially inserting the insert DNA in the opposite orientation desired. On the other hand, blunt ends are always compatible with each other.