Methods II

Question 0

Troubles working with DNA

Answer 0

• DNA is longer and harder to separate
• Genes are not discrete elements
• Solution: Restriction Endonuclease - cuts nucleus in the middle

Question 1

SDS-PAGE

Answer 1

• Gold standard for separating proteins
• This causes all the proteins to become Negatively charged and have the same Shape
• After this technique, proteins can be identified based on Size

Question 2

Sanger’s Dideoxy-Termination

Answer 2

• No Hydroxal group on these nucleotides
• Prevents Polymerase from adding nucleotides to the DNA chain
• Causes DNA fragments of different lengths
• Today we use robots to read a color and determine the base

Question 3

Libraries

Answer 3

Genomic Library
>DNA stored in bacteria cells for later use
>All containing identical genome
cDNA Library
>Specific to cell type, DNA will be different between cells
>Only stores genes that will be expressed at that time

Question 4

“Pulsed Field” Electrophoresis

Answer 4

*Electrophoresis where the electric field is reversed periodically

Question 5

Western Blots

Answer 5

To label DNA

• Radio or chemical labels during replication
• Probes (complimentary sequences)

Question 6

Cloning DNA

Answer 6

*Restriction Endonuclease produces Sticky ends on:
>Insert: ends to be cloned
>Plasmid: ends of Bacterium 
*Bacterium can only hold 1-30K bp
Solution: YAC or BAC