Cloning

Question 1

Restriction Enzyme Natural Function

Answer 1

cleaves foreign (unmethylated) DNA in the cell to protect the cell from invaders like HIV

Question 2

Restriction Enzyme Cut Site Properties

Answer 2

cleavage site is a palindrome, so it can be read the same way backwards and forwards

Question 3

Sticky Ends

Answer 3

what is at the end of the DNA after being cut with an endonuclease, overhangs of the DNA which can re-bond to each other

Question 4

Components of Cloning Plasmids

Answer 4

origin of replication to ensure replication of the plasmid
selectable marker for bacteria containing the plasmid (ex. antibiotic resistance)
restriction enzyme cleavage site
polylinker for multiple cleavage sites
strong promoter region
ribosome binding sequences

Question 5

cDNA Library

Answer 5

mRNA is copied by reverse transcriptase into DNA and inserted into a vector
represents only the expressed genes in a cell
only the coding region is present
number of copies related to expression level, long repetitive sequences all included
used as source of genetic information being put into expression vector

Question 6

Genomic Library

Answer 6

pieces of the genome are inserted into a vector such that all the clones represent the entire genome (intronic sequences, promoter regions, and long repetitive sequences all included)
used to study these other regions

Question 7

Producing Human Protein in Bacteria

Answer 7

choose gene of interest from a cDNA library, and amplify it using PCR, and put into a plasmid using the same restriction enzyme
insert DNA into the bacterial cell through transformation (CaCl2 or electroporation)
identify the bacteria that incorporated the plasmid through antibiotic selection
identify the cells that have a plasmid WITH the insert using blue white selection or reduction of antibiotic resistance
use the selected clones to amplify

Question 8

Bacterial Selection

Answer 8

a way of determining which of the bacteria have taken up the plasmid
antibiotic selection occurs when small molecules inhibit cellular enzymes, leading to cell death, but some bacteria that have taken the plasmid have been conferred with antibiotic resistance
also, antibiotic resistance can be used to determine if a bacteria has a plasmid containing the gene, by inserting the gene directly within the place for antibiotic resistance, stopping it
blue-white selection occurs in a similar way, in which a gene is inserted in the middle of the gene for betagalactosidase, stopping it from being made, therefore preventing the bacteria from having the ability to cleave gal into a blue substrate, leaving the colonies white

Question 9

Plasmid Digestion Map

Answer 9

week 10 worksheet

Question 10

Reporter Assay

Answer 10

replace the gene of interest with a reporter gene in order to study how the promoter for that gene works
use genomic library
reporter protein allows the amount to be easily measured

Question 11

Fusion Protein

Answer 11

GFP tag addd to another protein to be expressed, and therefor transcribed and translated to be attached to the original protein
allows us to visualize where certain proteins are expressed

Question 12

Yeast Two Hybrid Assay

Answer 12

two different proteins can interact to enhance transcription of a certain gene
two strains with the two proteins can mate and produce diploid cells
if you put these cells on a plate that requires large amount of this gene, you can isolate which ones mated

Question 13

Ligation

Answer 13

binding together two pieces of DNA (i.e. at their sticky ends)

Question 14

Transformation

Answer 14

inserting DNA into a cell where it can be replicated
methods include treating cells with calcium chloride (CaCl2) or electroporation (applying electric current to the membrane)