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34 Cards in this Set
- Front
- Back
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restriction enzyme
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cleave DNA
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Polymerases (DNA , thermostable, RNA)
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synthesize DNA or RNA
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Ligases
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join DNA
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RNases /Dnases/exonucleases
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degrade nucleic acids
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where does restriction enzyme cut?
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phosphodiester bonds of symmetry or palidromes
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Enzyme - kinases and phophatases
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phosphorylate or dephosphorylate proteins
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Enzyme - proteases
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degrade proteins
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Enzyme - Cellulases
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degrade starches
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Enzyme - Lipases
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degrade TAG's (fatty acids)
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How often to restriction enzymes cut DNA?
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4bp= 1/4+1/4+1/4+1/4=1/256
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Five facts about polymerases
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1. synthesize DNA/RNA
2.Require template 3.create dsDNA molecule 4. Taq used in PCR b/c can with stand denaturation stage |
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what are the requirements for a PCR reaction?
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1.thermocycler
2.template DNA 3.primers 4.polymerases 5.dNTP's 6.buffer |
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3 steps to PCR
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1.denaturation 94 dgrees C. breaks H-bonds
2.anneal primer.complementary homloguous pieces. 50-60 degrees C 3.Synthesis of new DNA 72 degrees C |
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At each cycle of the DNA how much is made?
How many runs is typical? How many copies can be created in a PCR run? |
Doubled
30 - 40 runs 2-4hrs 2 to the power of n= # of PCR cycles 30 copies = 1 billion copies |
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What are 3 types of vectors?
And what is the capacity of each? |
1.Plasmid, circular bacterial extrachromosmal DNA molecule - small capacity
2.Viruses, long term storage- bigger capacity 3.BACs and YACs, self replicating chroms-largest capacity |
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Describe the role of the plasmid/vector
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carry or transmit and/or replicate or synthesize DNA/RNA/protein
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What features do all plasmids require?
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1.cloning site
2.replication origin ( can have more than one) |
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What do selection genes procedure accomplish?
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produce a protein that detoxifies a selection agent that prevents growth of cells that don't contain the plasmid.
another method is color screening |
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Multiple cloning site (MCS)
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multiple restriction enzyme cut sites
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Colour screening
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allows for selection of cells that contain and insert in the plasmid
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expression vectors
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permits translation of cloned DNA in host cell
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3 steps to cloning
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1.DNA of interest is cut with one or two restriction enzymes
2.vector DNA is cut with same enzyme 3.digested vector and DNA of interest are mixed and DNA ligase is added |
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name a way to select for growth of cells that contain the plasmid?
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use antibiotic resistance genes in plasmid
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once ligated the vector is mixed with host cells. this is called?
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transformation
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chain termination is used for what procedure?
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sequencing of DNA
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what is added to the 4 tubes for this process?
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4 dNTPs,
primer DNA polymerases fluorescent ddNTP (dideoxy base) |
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what makes mitochondrial DNA unique?
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semi-autonomous organelle
energy factory own genome maternally inherited high mutation rate useful for genetic comparisons |
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name 4 blotting techniques
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southern-transfer of DNA to membrane
northern-transfer of RNA western-transfer of proteins eastern-transfer of other |
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describe blotting
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transfer of a macromolecule to a solid substrate after electrophoresis
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how does electrophoresis separate?
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size or charge
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how is the transfer accomplished?
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salt, vacuum, or electric current
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how is a blot analylized?
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using hybridization (labeled probe of molecule you are wishing to identify)
after washing only the labeled probe remains bound to the target |
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what is a probe made of?
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radioactive nucleotide sometimes a antibody
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uses of blotting (3)?
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1.DNA-copy number, allelic variation determination
2.RNA-gene expression patterns 3.Protein-gene expression patterns, protein structure |
