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 |
