BIO 357- Exam 2
Describe how you would clone a specific DNA of interest into a plasmid DNA vector -
answerThe steps in the DNA cloning process are to isolate the gene of interest and cut
it and a plasmid vector with restriction enzymes. The gene of interest and the plasmid
are then ligated together with DNA ligase. Then, the recombinant DNA can be inserted
into a cell for replication.
Be able to explain a strategy to identify a bacterial colony carrying a recombinant
plasmid DNA if you are inserting your gene of interest into the ampicillin-resistance
gene on the plasmid pBR322. - answerCells containing recombinant plasmids can often
be identified as containing recombinant plasmids by screening for the insertional
inactivation of a second genetic marker on the plasmid.
What is selection? - answerA strategy to set a condition to select away things that you
DONT want or for the things that you DO want
Auxotroph = mutant that cannot grow on minimal medium, requires certain
supplement(s).
Prototroph= wild type, it will grow in minimal medium or medium lacking the supplement.
What is screen? - answerA Strategy to use different criteria to screen through
transformants to FIND what you want
Use Human cDNA library
To screen for cDNA clones that can rescue fission yeast cdc2 mutant.
For one type of screen, how can one find something (cells) that will die on Ampicillin? -
answerpBR322 (Plasmid 1) is 4.3 kb long.
- Bacterial cells carrying this plasmid can grow on the media containing both ampicillin
and tetracycline.
Digest with Pst1 (cuts)
- Pst1 cut will interrupt the Ampr gene (Ampr encodes a protein that can degrade
degrade ampicillin)
Bacterial cells carrying this plasmid can grow on media containing tetracycline, not
ampcillin!
What is a yeast auxotroph and how auxotrophs can be in selections in yeast two-hybrid
, assay? - answerYeast auxotroph: a yeast strain that requires a particular addition
nutrient which the normal strain does not.
Genes can supply nutrient: HIS- , LEU-, TRY-, HIS3, LEU2, TRY1
The yeast 2-hybrid (Y2H) assay is a well-established technique to detect protein-protein
interactions. This is an extremely powerful tool for researchers and is often used
alongside one or two other methods to examine the multitude of interactions that take
place in cells.
two-hybrid assay - answerworks by requiring an interaction between two proteins,
where one has a DNA-binding domain and the other has a transcription-activation
domain
What and how restriction enzymes are used in recombinant DNA technology? -
answerRestriction Enzymes: Enzymes that cut DNA at a specific sequence of
nucleotides.
The first step in the development of recombinant DNA technology was the
characterization of restriction endonucleases—enzymes that cleave DNA at specific
sequences. These enzymes were identified in bacteria, where they apparently provide a
defense against the entry of foreign DNA (e.g., from a virus) into the cell.
What screen strategies can you use to identify your desired recombinant plasmid?
(antibiotic-resistance, plasmid prep and restriction digestion, PCR screening) -
answerAntibiotic resistance: it allows a scientist to easily detect plasmid-containing
bacteria when the cells are grown on selective media and provides those bacteria with
pressure to keep your plasmid.
Plasmid Prep: isolate small plasmid DNA from bacteria while limiting contaminating
proteins and genomic DNA. The plasmid quality is acceptable for restriction analysis,
sequencing, cloning, or other purposes, but should not be used without additional
cleanup for embryonic injections.
Restriction digestion: Cells containing recombinant plasmids can often be identified as
containing recombinant plasmids by screening for the insertional inactivation of a
second genetic marker on the plasmid.
PCR screening: The PCR technique produces multiple copies of a particular DNA
sequence in vitro through repeated cycles of PCR reactions. Recombinant DNA is
produced in order to locate the gene. Recombinant DNA is not produced.
- 1) Pick a colony, 2) replica plate, 3) Add PCR master mix. 4) Perfrom PCR - Run PCR
samples on DNA gel
Describe how PCR works. Describe the required components in a PCR reaction.
Explain
Describe how you would clone a specific DNA of interest into a plasmid DNA vector -
answerThe steps in the DNA cloning process are to isolate the gene of interest and cut
it and a plasmid vector with restriction enzymes. The gene of interest and the plasmid
are then ligated together with DNA ligase. Then, the recombinant DNA can be inserted
into a cell for replication.
Be able to explain a strategy to identify a bacterial colony carrying a recombinant
plasmid DNA if you are inserting your gene of interest into the ampicillin-resistance
gene on the plasmid pBR322. - answerCells containing recombinant plasmids can often
be identified as containing recombinant plasmids by screening for the insertional
inactivation of a second genetic marker on the plasmid.
What is selection? - answerA strategy to set a condition to select away things that you
DONT want or for the things that you DO want
Auxotroph = mutant that cannot grow on minimal medium, requires certain
supplement(s).
Prototroph= wild type, it will grow in minimal medium or medium lacking the supplement.
What is screen? - answerA Strategy to use different criteria to screen through
transformants to FIND what you want
Use Human cDNA library
To screen for cDNA clones that can rescue fission yeast cdc2 mutant.
For one type of screen, how can one find something (cells) that will die on Ampicillin? -
answerpBR322 (Plasmid 1) is 4.3 kb long.
- Bacterial cells carrying this plasmid can grow on the media containing both ampicillin
and tetracycline.
Digest with Pst1 (cuts)
- Pst1 cut will interrupt the Ampr gene (Ampr encodes a protein that can degrade
degrade ampicillin)
Bacterial cells carrying this plasmid can grow on media containing tetracycline, not
ampcillin!
What is a yeast auxotroph and how auxotrophs can be in selections in yeast two-hybrid
, assay? - answerYeast auxotroph: a yeast strain that requires a particular addition
nutrient which the normal strain does not.
Genes can supply nutrient: HIS- , LEU-, TRY-, HIS3, LEU2, TRY1
The yeast 2-hybrid (Y2H) assay is a well-established technique to detect protein-protein
interactions. This is an extremely powerful tool for researchers and is often used
alongside one or two other methods to examine the multitude of interactions that take
place in cells.
two-hybrid assay - answerworks by requiring an interaction between two proteins,
where one has a DNA-binding domain and the other has a transcription-activation
domain
What and how restriction enzymes are used in recombinant DNA technology? -
answerRestriction Enzymes: Enzymes that cut DNA at a specific sequence of
nucleotides.
The first step in the development of recombinant DNA technology was the
characterization of restriction endonucleases—enzymes that cleave DNA at specific
sequences. These enzymes were identified in bacteria, where they apparently provide a
defense against the entry of foreign DNA (e.g., from a virus) into the cell.
What screen strategies can you use to identify your desired recombinant plasmid?
(antibiotic-resistance, plasmid prep and restriction digestion, PCR screening) -
answerAntibiotic resistance: it allows a scientist to easily detect plasmid-containing
bacteria when the cells are grown on selective media and provides those bacteria with
pressure to keep your plasmid.
Plasmid Prep: isolate small plasmid DNA from bacteria while limiting contaminating
proteins and genomic DNA. The plasmid quality is acceptable for restriction analysis,
sequencing, cloning, or other purposes, but should not be used without additional
cleanup for embryonic injections.
Restriction digestion: Cells containing recombinant plasmids can often be identified as
containing recombinant plasmids by screening for the insertional inactivation of a
second genetic marker on the plasmid.
PCR screening: The PCR technique produces multiple copies of a particular DNA
sequence in vitro through repeated cycles of PCR reactions. Recombinant DNA is
produced in order to locate the gene. Recombinant DNA is not produced.
- 1) Pick a colony, 2) replica plate, 3) Add PCR master mix. 4) Perfrom PCR - Run PCR
samples on DNA gel
Describe how PCR works. Describe the required components in a PCR reaction.
Explain
