BIOTECH EXAM 2 COMPLETE STUDY GUIDE
WITH SOLVED QUESTIONS
◉ 1 to 4 kb.
Answer: How small is Plasmid DNA?
◉ Vectors.
Answer: Pieces of DNA that can accept, carry, and replicate other
pieces of DNA.
◉ Calcium Chloride Transformation of Bacterial Cells.
Answer: A process for inserting foreign DNA into bacteria.
(ineffective)
◉ What is the 1st step?.
Answer: Treat bacterial cells with calcium chloride while chilled on
ice for 30 minutes.
◉ What is the 2nd step?.
Answer: Add plasmid DNA to cells chilled on ice.
◉ What is the 3rd step?.
,Answer: Heat the cell and DNA mixture (30 seconds at 42 degrees
Celsius)
◉ What is the 4th step?.
Answer: Plasmid DNA enters bacterial cells and is replicated and
express their genes.
◉ Electroporation.
Answer: applies a brief pulse of high voltage electricity that creates
tiny holes in the cell wall that allows DNA to enter.
◉ What are advantages of electroporation?.
Answer: Rapid, requires few cells, can be use to introduce DNA into
other cell types, more efficient process.
◉ Selection.
Answer: process that facilitates the identification of recombinant
bacteria from bacteria that contain plasmid without foreign DNA,
while preventing the growth of non-transformed bacteria.
◉ Antibiotic selection.
Answer: transformed cells are cultured on plates containing
antibiotics to identify recombinant bacteria and non-transformed
bacteria
,◉ What does Antibiotic selection NOT select?.
Answer: plasmid containing foreign DNA vs. recircularized plasmid
with no DNA insert.
◉ Blue-white selection with X-gal in media.
Answer: DNA is cloned into plasmid restriction site within LacZ
gene.
◉ What happens when Blue-white selection is interrupted by an
inserted gene?.
Answer: the laxZ gene cannot produce functional Beta galactosidase.
◉ What is non-functional lacZ?.
Answer: White colony. Clones. Genetically identical bacterial cells
each containing copies of recombinant plasmid.
◉ What happens when X-gal is added to media in petri plate?.
Answer: functional lacZ=blue colony=clones= genetically identical
bacterial cells each containing recircularized plasmid. no transgene.
◉ What was the first recombinant human protein marketed?.
Answer: Insulin, 1982
, ◉ What was the next human protein marketed?.
Answer: growth hormone, 1985
◉ What was source of growth hormone prior to recombinant
technology?.
Answer: cadavers
◉ What makes a good vector in regards to size?.
Answer: small enough to be separated from chromosomal DNA of
host plasmid
◉ What makes a good vector in regards to origin of replication?.
Answer: site for DNA replication that allow plasmids to replicate
independently from host chromosome
◉ What is copy number?.
Answer: number of plasmids in the cell; normally small
(recombinant plasmids have high copy numbers)
◉ What makes a good vector in regards to Multiple cloning site?.
Answer: recognition sites for several restriction enzymes in which
DNA insert is cloned into
WITH SOLVED QUESTIONS
◉ 1 to 4 kb.
Answer: How small is Plasmid DNA?
◉ Vectors.
Answer: Pieces of DNA that can accept, carry, and replicate other
pieces of DNA.
◉ Calcium Chloride Transformation of Bacterial Cells.
Answer: A process for inserting foreign DNA into bacteria.
(ineffective)
◉ What is the 1st step?.
Answer: Treat bacterial cells with calcium chloride while chilled on
ice for 30 minutes.
◉ What is the 2nd step?.
Answer: Add plasmid DNA to cells chilled on ice.
◉ What is the 3rd step?.
,Answer: Heat the cell and DNA mixture (30 seconds at 42 degrees
Celsius)
◉ What is the 4th step?.
Answer: Plasmid DNA enters bacterial cells and is replicated and
express their genes.
◉ Electroporation.
Answer: applies a brief pulse of high voltage electricity that creates
tiny holes in the cell wall that allows DNA to enter.
◉ What are advantages of electroporation?.
Answer: Rapid, requires few cells, can be use to introduce DNA into
other cell types, more efficient process.
◉ Selection.
Answer: process that facilitates the identification of recombinant
bacteria from bacteria that contain plasmid without foreign DNA,
while preventing the growth of non-transformed bacteria.
◉ Antibiotic selection.
Answer: transformed cells are cultured on plates containing
antibiotics to identify recombinant bacteria and non-transformed
bacteria
,◉ What does Antibiotic selection NOT select?.
Answer: plasmid containing foreign DNA vs. recircularized plasmid
with no DNA insert.
◉ Blue-white selection with X-gal in media.
Answer: DNA is cloned into plasmid restriction site within LacZ
gene.
◉ What happens when Blue-white selection is interrupted by an
inserted gene?.
Answer: the laxZ gene cannot produce functional Beta galactosidase.
◉ What is non-functional lacZ?.
Answer: White colony. Clones. Genetically identical bacterial cells
each containing copies of recombinant plasmid.
◉ What happens when X-gal is added to media in petri plate?.
Answer: functional lacZ=blue colony=clones= genetically identical
bacterial cells each containing recircularized plasmid. no transgene.
◉ What was the first recombinant human protein marketed?.
Answer: Insulin, 1982
, ◉ What was the next human protein marketed?.
Answer: growth hormone, 1985
◉ What was source of growth hormone prior to recombinant
technology?.
Answer: cadavers
◉ What makes a good vector in regards to size?.
Answer: small enough to be separated from chromosomal DNA of
host plasmid
◉ What makes a good vector in regards to origin of replication?.
Answer: site for DNA replication that allow plasmids to replicate
independently from host chromosome
◉ What is copy number?.
Answer: number of plasmids in the cell; normally small
(recombinant plasmids have high copy numbers)
◉ What makes a good vector in regards to Multiple cloning site?.
Answer: recognition sites for several restriction enzymes in which
DNA insert is cloned into
