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42 Cards in this Set

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vector means the same thing as …
replicon
DNA cloning means the same as …
molecular cloning
foreign DNA means the same as … or … DNA
insert
target
None
what are the 4 essential steps in cell-based cloning?
1. Formation of recombinant DNA
2. Transformation
3. Amplification to produce numerous cell clones
4. Isolation of recombinant DNA clones
None
how do you form a recombinant DNA?
separately digest purified DNA and vector DNA and with restriction endonuclease and then ligate together
describe step 2 - transformation:
introduce recombinant DNA into bacterial cells … the cells only take up one recombinant DNA
describe step 3 - amplification:
the transformed bacterial cells are plated and then individual colonies are picked up and grown in nutrient media
what is the 4th step in DNA cloning in bacterial cells?
isolation of recombinant DNA clones
… cleaves DNA at a recognition sequence
restriction enzyme/endonuclease
restriction enzyme recognition sequences have the properties of being …
palindromic
usually 4-6 bp in length
None
the frequency of restriction sites on a DNA molecule depends on the …
length of the recognition site
DNA cleavage by restriction enzymes can produce either … or … ends
blunt
sticky
None
what are the 2 properties that a cleaved DNA has due to the sequence being palindromic:
cleaved ends are complimentary
two strands have same nucleoide sequence running 5' to 3'
None
a. EcoRI
b. SmaI
identify the restriction enzyme belonging to the identified recognition site:
a.
b.
what is the function of DNA ligases?
rejoins sticky or blunt ends by reforming the phosphodiester bond
a restriction enzyme with a recognition site that is 4 bps long will cut every … bases
4^4 (258)
a restriction enzyme with a recognition site that is 6 bps long will cut every … bases
4^6 (4096)
… is used to separate digested DNA according to it's size
gel electrophoresis
migration through the gel matrix in an electric field is influenced by … and … and …
size
charge
shape
None
the overall charge of DNA is …
negative
larger DNA fragments will migrate (faster/slower) than smaller fragments in gel electrophoresis
slower
what is the base pair difference that gel electrophoresis can detect?
1 bp
positive
negative
Anode: an electrode from which electrons flow out of create a … charge
Cathode: an electrode in which electrons flow into creating a ... charge
None
vector DNA is also know as a …
plasmid
plasmids are extrachromosomal elements that:
a. incorporate themselves in host DNA
b. replicate autonomously
None
the larger the insert size of the DNA the (lower/higher) the overall yield
lower
the hybrid - the combination of the vector and the insert (foreign) DNA is also known as …
recombinant DNA
heat shock is a method used for … (used to temporarily disrupt the cell membrane of the host bacteria
transformation
competence is …
the ability of a cell to take up DNA from the environment
PCR stands for …
polymerase chain reaction
what are the 4 ingrediants for PCR?
1. DNA sample to be amplified
2. complementary primars for 3' flanking regions of your target DNA (in excess)
3. Deoxyribonucleotides (in excess)
4. Heat-stable DNA polymerase
None
which DNA polymerase lacks 3' to 5' exonuclease activity and has a low fidelity?
taq (thermus aquaticus)
which DNA polymerase has 3' to 5' exonuclease activity and has a high fidelity?
pfu (pyrococcus furiosus)
PCR has 3 series of successive reactions, what are they?
1. denaturation of DNA
2. annealing of primer
3. DNA synthesis
why would one want to clone a PCR product?
the amount of DNA from PCR reaction is limited
what is T-A cloning?
taq leaves a 3' 'A' overhang in PCR products and the products can be ligated into vectors with 5' 'T' overhangs
pfu creates (sticky/blunt) end clones
blunt
briefly describe a southern blot:
DNA is electorphoresed and transferred to a membrane. DNA clone is then used as a hybridization probe against target DNA from gel
briefly describe a northern blot:
RNA is electorphoresed and transferred to a membrane. a hybridization probe is then used to detect target RNA.
briefly describe a western blot:
proteins are size fractionated on a polyacrylamide gel and then probed with an antibody
a working form of a hybridization probe must be ... stranded and must be ...
single
labeled (radioactive or fluorescence)
put the following steps in order:
a. Separate DNA digests by electrophoresis
b. Expose membrane to x-ray film
c. Digest with restriction endonuclease
d. Transfer DNA from gel to membrane
e. Hybridize membrane with probe
c, a, d, e, b