Molecular Biology/Genetics MIC 445 – Flashcards

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Kary Mullis
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Developed the Polymerase Chain Reaction (PCR)
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Central Dogma
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DNA->RNA->Protein
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Replication
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DNA Synthesis
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Arthur Kornberg
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Discovered the function of DNA Polymerase
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5'-3'
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Direction of DNA synthesis, RNA Polymerase
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Semiconservative Repication
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Method of DNA Strand Templating, each helix contains a parental strand and a daughter strand
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Semidiscontinous Strand Growth
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One strand is replicated continuously in the direction of the movement of the replicating fork.
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Okasaki Fragments
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Located in the lagging strand that is replicated discontinously
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RNA Primers (5'-3')
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Are needed for initiation of DNA synthesis
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Primase
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Binds directly to DNA without help from nucleotides, synthesizes primer to initiate DNA synthesis
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Bi-directional Replication
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Occurs in Prokaryotic and Eukaryotic Cells (exceptions are bacterial plasmids and linear DNA viruses)
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Bi-Directional Forks
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Are created by chromosomal replication
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100 Base Pairs/Second
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The rate of fork movement in human cells.
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10,000 to 100,000
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Possible locations of fork formation in the human genone
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Specific Chromosomal Sites
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Where DNA replication begins
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Helicase (DnaB)
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Melts the two strands of the chromosome to generate unpaired template strands
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DNA Pol III
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Synthesizes the leading strand
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Multiple primers, 2 nucleases, DNA pol, and Ligase
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Are required to develop the lagging strand of the replication fork
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DNA replicases
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DNA pols that make new double stranded DNA's (dsDNA)
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Replicase
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de novo synthesis of new strands of DNA; catalyzes chain elongation at the growing fork
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Telomeres
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Consist of repetitive oligometric sequences, are needed because the lagging strand is copied in discontinuously which would create a problem for linear DNA.
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rNTP
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Ribonucleotides with three phosphate groups, is the building block of RNA synthesis and synthesis of primers in DNA replication
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Accuracy of Transcription
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Is not as accurate as replication
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Only one strand is copied
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In Transcription
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RNA pol does not require a________to initiate
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primer
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rRNA
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Form the core of ribosomes
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mRNA
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Used in translation to make protein
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tRNA
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Adaptors that link amino acids to mRNA during translation
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snRNA
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RNA splicing of pre-mRNA to mRNA, found in nucleus of eukaryotic cells
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srRNA
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Non-coding DNA
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Stable ribosomal RNA has
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Three characteristic molecular weights
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Rate of synthesis in transcription
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50 nucleotides/sec/molecule
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Complementary copy of the template strand
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mRNA in Transcription
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Leader Sequence
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5',25-200 nucleotides with a ribosome binding sequence
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AUG
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Translation Start Codon
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UAG, UAA, or UGA
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Translation stop codon
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Introns in eukaryotes
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Intervening sequences that are removed prior to translation
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3 RNA Polymerases
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in Eukaryotes
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Single RNA polymerase
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In Prokaryotes
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5'-Ribonucleoside triposphates
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Are used to synthesize mRNA
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Initiation
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When RNA pol docks at a promoter, first step of transcription
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Elongation
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RNA chain elongates, second step of transcription
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Termination
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Pol reaches a terminator and releases the completed transcript, third and final step of transcription
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The promoter is always located
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Upstream of the gene
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Promoter
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DNA sequence that the RNA pol binds to initially.
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Transcription start site is also called the
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Initiation site
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Predominant mechanism that the cell uses to control what proteins will be made at a given time
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Initiation of transcription
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Promoter strength depends on
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Similarity to consensus sequence
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Mechanism of control of transcription initiation rates
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Divergent sequences
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The most common bacterial promoters are located at
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-35 and -10
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Closed Promoter Complex
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Known as the promoter sequence in duplex DNA, RNA Pol Core binds to this first in initiation.
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Elongation
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Polymerase advances 3'-5' down template strand, melting duplex DNA and adding rNTP's to growing DNA
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Operons
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Mechanism used by bacteria to control gene expression, share a single promoter
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lacZ, lacY, lacA
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Are genes located in the lac operon, are transcribed together
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Core Promoter
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Where polymerase binds
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RNA Pol II
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Transcribes genes into mRNA's in nucleus
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RNA Pol III
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Transcribes tRNA's, 5S rRNA's and snRNA's, genes transcribed by this can carry promoter sequences deep within the coding region
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RNA Pol I
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Located in the nucleus, transcribes all rRNA genes except for 5SrRNA
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Translation
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Most highly conserved process, most energy cost
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Features of the genetic code relevant to translation
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Codons are sets of 3 bases in the mRNA, codons instruct ribosomes to incorporate specific amino acids into the polypeptide chain, code is non-overlapping and gapless, 61 codons direct the incorporation of 20 a.a.
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Proteins are synthesized from
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amino (N) terminus to carboxyl (C) terminus
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Peptide bonds are synthesized at
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15 amino acids/second, same rate of transcription
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Coupled transcription/translation
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Occurs in Prokaryotes
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Polysomes are formed when
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Multiple ribosomes translate a mRNA at the same time
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Non standard base pairing can occur between
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Anti-codon and Codon, wobbles occur at the 3rd codon position or 1st anti-codon position
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Anticodon-Codon base pairing is
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Imprecise due to wobble and isoaccepting tRNA's
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fMet-tRNA
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Initiates prokaryotic protein synthesis
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tRNAf and tRNAm
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Recognize the AUG codon
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aminoacyl-tRNA synthase
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Charge tRNAf and tRNAm with (Met)
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The first amino acid at the amino terminus of the polypeptide chain
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fMet-tRNAf
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Met-tRNAm
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Use at internal positions of the polypeptide chain, used in elongation
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Met-tRNAi
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Used to start protein synthesis
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Ribosomal subunits that disassemble after each round of translation
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(30S/40S), (50S, 60S)
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Initiation Complex
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Formed when a free 30S/40S subunit combines with an mRNA translation site + fMet-tRNAf or Met-tRNAi
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70S/80S ribosome
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Consists of an AUG codon and an attached 70S/80S ribosome
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Release factors
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Are required to terminate protein synthesis, release the polypeptide chain from the last tRNA, and release the ribosome from the mRNA
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Elongation factor (EF-G/EF-2)
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Translocates the growing chain with it's mRNA to P site, final step of Elongation
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Elongation factor (EF-G/EF-2)
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Translocates the growing chain with it's mRNA to P site, final step of Elongation
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Restriction-modification system in bacteria consists of
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Restriction endonucleases and methylases.
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Palindromic sequences
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Sequences that are the same but in alternating directions, sites of methylation.
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If the DNA is properly methylated
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Specific nucleases cannot cleave the recognition sequence.
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EcoRi
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"Six cutter", leave 5' overhang.
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Alu I
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"Four cutter", leaves blunt ends to the DNA
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Bgl
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"Six cutter with interrupted palindrome". Leaves 5' overhang
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Aat II
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"Six cutter" with 3' overhang. Same recognition sequence as Bsa HI, but different cleavage position
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The utility of restriction endonucleases lies in their
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Sequence specificity and the relatively predictable frequency with which the recognition sites occur within any DNA sample
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If there is a 25% probability for a specific base at any given site, the frequency with which different restriction endonuclease sites will occur will be
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0.25^N, N being the length of the recognition site
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Exonuclease III
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Catalyzes the stepwise removal of nucleotides from the 3' terminal of duplex DNA
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Mung Bean Nuclease
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Single strand specific DNA and RNA nuclease which will degrade single strand extensions from the ends of DNA and RNA and leave blunt ends
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Ligase
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Catalyze the formation of a phosphodiester bond between juxtaposed 5' phosphate and 3' hydroxyl termini of nucleotides. Act as paste for restriction nucleases.
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T4 Polynucleotide Kinase
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Catalyzes the transfer and exchange of a phosphate group between the gamma position of rATP to the 5'OH terminus of ds/ss DNA/RNA. Also removes 3'P groups.
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Calf Intestinal Phosphatase
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Catalyzes the removal of 5' phosphate groups from RNA and DNA. Treated DNA cannot self-ligate
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Endogenous (Natural) Plasmids
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Self-replicating extrachromosomal DNA, contain mechanisms to maintain a stable copy number in their bacterial hosts and partition molecules accurately to daughter cells.
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If the molar ration of insert DNA to plasmid vector is too high or two low, then
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Plasmids carrying tandem inserts of the DNA fragment or empty plasmids will be generated
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All cloning vectors contain
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Origin of replication/replicon, Antibiotic resistance/selectable marker, multiple cloning site
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Plasmid replicon
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Smallest piece of DNA that is able to replicate autonomously and maintain it's copy number
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Critical Factors in Electroporation
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Conductivity (NaCl present), Field Intensity, Pulse Length, Temperature (cold)
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Transient Pores
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Created by chemical and physical methods, allows DNA to pass through the cell
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Methods of Transformation
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Electroporation, or Heatshock/Divalent Cation
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Competency
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Appropriate physiological state that allows for chemical transformation
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Role of Ca++ in treatment of bacteria on ice
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Crystallizes fluid membranes, stabilizes distribution of charged molecules in membrane
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Role of Cl2 in treatment of bacteria on ice
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Causes the cells to swell with water, necessary for DNA uptake
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Role of heatshock in CaCl2 protocol
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Increases permeability of cell membrane
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LacZ Gene
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Encodes for beta-galactosidase, serves as an inductible reporter gene
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beta-galactosidase converts X-Gal into
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Galactose and blue indigo
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1 OD Unit =
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0.8 x 10^9 cells/ml
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Stationary phase is at a density of
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2-3 x 10^9 cells/ml
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Phenol
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Protein denaturant, facilitates protein removal from nucleic acids
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Chloroform
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Protein denaturant, stabilizes interphase, increases density of mixture, removes lipids
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Isoamyl alcohol (IAA)
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Prevents foaming of phenol/chloroform mixes during vortexing, enhances phase separation
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DNA Purification at pH<7
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DNA denatures into organic phase, RNA in aqueous phase
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DNA purification pH>7
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DNA and RNA partition in aqueous phase
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The purpose of adding salts to the precipitation
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To neutralize the negative charge of DNA
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The purpose of adding ethanol to DNA precipitation
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To remove water, increases electrostatic force between ions, facilitates formation of ion pairs that results in efficient precipitation of DNA
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Isoaccepting tRNAs
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More than 1 tRNA molecule can be charged with the same amino acid
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Aminoacyl-tRNA Synthetases
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Activate amino acids and tRNA's by covalently linking them (charging tRNA), has effective proofreading activity
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What feature of Replication makes telomeres necessary?
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Discontinous synthesis of the lagging strand
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Telomerase
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Is a reverse transcriptase that serves to extend the 3' end of the lagging strand at a telomere during eukaryotic replication
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Features common to cloning vectors
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Origin of replication, selectable marker, multiple cloning site
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