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40 Cards in this Set

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what are the 4 basic ideas of recombinant DNA technology?
-isolation
-amplification
-characterization
-alteration = manipulation
a replicon is also known as a ...
vector
DNA cloning = ... cloning
molecular
4 essential steps of cell-based cloning:
-... of recombinant DNA
-...
-... to produce numerous cell clones
-... of recombinant DNA clones
formation
transformation
amplification
isolation
Step 1: formation of recombinant DNA
-purified ...
-purified ...
-separately digest with a ...
-add the digested component together and ...
-result is ...
DNA
vector
restriction endonuclease
ligate
recombinant DNA molecule
Step 2: transformation
-... the recombinant DNA molecules into the bacterial cell
-Key step, as the cells normally take up only # recombinant DNA molecule
-... or recombinant molecule occurs
introduce
1
replication
Step 3: Amplification to produce numerous clones
-the individual clone colonies are separated out on a ... dish for growth
-these colonies are then individually picked into a ... step (homogeneity)
nutrient agar
secondary amplification
Step 4-
... of recombinant DNA clones
isolation
What will you need?
-Enzymes: ...(scissors), ...(glue), ... (for PCR)

-... (vehicles)

-... (source DNA from which you will create a genomic library, cDNA library or simply a clone)
restriction enzymes
ligases
DNA polymerases

vectors

Host cells
restriction endonucleases (or restriction enzymes)
-cut double stranded DNA at a very specific ...
-recognize specific ... sequences
-produces either overhangs (... ends) or ... ends
-cleave DNA at specific sequences regardless of their ...
restriction site
palandromic
sticky, blunt
origin
Any 2 fragments of DNA produced by the same restriction enzyme can be joined together because DNA ligase rejoins sticky or blunt ends by reforming the ... bond
phosphodiester
what can you use to separate DNA fragments?
gel electrophoresis
migration through the gel matrix in an electric field is influenced by what 4 factors?
size
shape
charge
chemical composition of molecule
DNA and RNA migrate towards the positive end (...)
anode
why separate by electrophoresis?
-visual difference in fragment ... difference
-... of DNA fragment
-further analysis by ...
length
purification
Southern blotting
what is an electrode from which electrons flow out of creating a positive charge?
anode
what is an electrode in which electrons flow into creating a negative charge?
cathode
vector-plasmids
-naturally occurring extrachromasomal elements that ... autonomously (at high numbers)
-high copy numbers (amplification)
-selection (stably confer antibiotic ... to host)
replicate
resistence
What is this?
-DNA 'vehicle" that is able to replicate and maintain itself within a host cell
-contains polylinkers with restriction sites not found elsewhere in the plasmid
-used to carry insert DNA (up to 5kb); confers similar properties to insert DNA
-selectable by some phenotype to identify host cells which carry this
vector
The larger the insert size, the ... the overall yield of insert
lower
After a restriction enzyme cleaves the vector at the unique restriction site, the DNA inserts with compatible ends to the restriction site and is ligated via ligase into the vector.
The result is ... or ... (hybrid between the vector and the insert (foreign) DNA)
recombinant DNA
chimera
The next step is to get the recombinant DNA into a host for ... (transformation)
amplification
what is the process in which there occurs an uptake of naked DNA from the environment by a bacterial cell?
transformation
The host is used to grow your hybrid or chimera in to ensure rapid ... and high ...
growth
yields
2 different types of amplification:
-... bacterium replicates
-... replicates within the host (up to 500 plasmids)
host
plasmid
What is the ability of a cell to take up DNA from the environment (temporary disruption of cell membrane)

-you can do this through 2 ways, ... (heat shock) or ...
competence

chemical treatment
electrical stimulation
selection
-transformed cells are ... selection
-recombinant cells are ... inactivation (visual selection)
antibiotic
insertional activation
what is this?
-selective, exponential amplification from a heterogeneous population of DNA
-rapid, easy, sensitive, robust
polymerase chain reaction (PCR)
downsides to PCR
-sequence information (no longer a problem-HGP
-fragments usually ...
-the longer the fragment, the less ...
short
amplified
requirements for PCR:
-DNA
-primers (in excess)
-deoxyribonucleotides (in excess)
-heat stable DNA polymerase (taq polymerase, which is lacks 3'-5' exonuclease activity and has... fidelity(error prone); and Pfu polymerase, which has 3'-5' exonuclease activity and ... fidelity (low error rates)
low
high
PCR has a cyclic series of what 3 successive reactions?
-... of DNA
-primer ...
-DNA ...
denaturation
annealing
synthesis
We ... a PCR product because it provides us with large quantities for proper analysis. The amount of DNA material produced from a PCR reaction is limited
clone
what type of cloning is this?
-Taq polymerase leaves a 3' A overhang on PCR products
-ligated into vectors that have 5'T overhangs
T-A cloning
what type of cloning is this?
-polishing enzymes, including the Pfu polymerase, create blunt ends
bunt end cloning
what is this?
base pairing between the probe and the target
hybridization
what is this?
-complex mixture of unlabeled nucleic acid molecules
-in molecular cloning refers to specific DNA fragments (amplify)
Target DNA
what is this?
-labeled nucleic acid (single-stranded of double-stranded)
-working form must be single-stranded for base-pairing to occur
-labeled with radioactivity or fluorescence
probe
how do you remember what blot does what?
SNOW DROP
-southern --> DNA
-northern --> RNA
-western --> protein
after size fractionation by gel electrophoresis, the DNA is denatured and transferred to a membrane for hybridization, this is ...
southern blot
what are the 5 steps to blotting?
-restriction enzyme digestion
-gel electrophoresis
-transfer
-hybridization
-detection