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BMES-219
week1
| Question | Answer |
|---|---|
| Aliivibrio fischeri (A. fischeri) | Gram negative, rod shaped bacteria predominantly found living in symbiosis with a diverse array of marine organisms. It’s chromosomal DNA (chDNA) contains the lux operon, which codes for proteins that are necessary for bioluminescence. |
| Escherichia coli (E. coli) | Gram negative, rod shaped bacteria. Commonly found in the lower gut in warm-blooded animals. Prokaryotic model organism for biotechnology and microbiology as the host for working with recombinant DNA. |
| Gram-Negative Bacteria | External LPS layer (lipopolysaccharide). Single Layer of peptidoglycan based cell wall. Both A. fischeri and E. coli are Gram-negative |
| Operon | genetic regulatory system in which genes coding for functionally related proteins are clustered along the DNA. This feature allows protein synthesis to be controlled coordinately in response to the needs of the cell. |
| Chromosomal DNA | large circular piece of DNA (Bacteria). For our project the chromosomal DNA comes from A. fischeri and we will be isolating the lux operon from this. |
| Restriction Digestion | enzymatic reactions that cut DNA into smaller pieces. Mediated by enzymes that will digest the DNA at only specific sequences |
| Plasmid | small circular DNA used to move DNA from one organism into a second; vector |
| Ligation | enzymatic reaction that will “paste” two pieces of DNA together. Pieces can be from the same organisms or two different sources |
| Recombinant DNA | Term for what is produced when DNA from two different sources (chDNA + Plasmid DNA) |
| Shotgun Cloning | is the practice of randomly digesting a large piece of DNA to smaller pieces that can then be ligated into plasmids for transport to other organisms. |
| Genomic Library | collection of cloned DNA fragments from a genome each part of the genome is represented in the library. it can be screened for sequences of interest. In our project the genome is from A. fischeri and the sequence of interest is the lux operon . |
| Clone | A bacteria housing a recombinant plasmid that contains a piece of genomic DNA; it doesn’t have to contain the gene you are looking for |
| Resuspension Buffer (Buffer ATL) | contains sodium dodecyl sulphate (SDS) a strong anionic detergent that can solubilize the membrane proteins. The detergent disrupts the lipid bilayer of gram-negative cells and brings the proteins into solution as protein-lipid-detergent complexes |
| Proteinase K | an enzyme that will aid in the release of nucleic acids while deactivating nucleases (enzymes that degrade nucleic acids) present. |
| RNAse | RNase A is an efficacious ribonuclease that is used to degrade RNA that is present in the sample. It cleaves the 3’ side of phosphodiester bonds after pyrimidine nucleotides in single stranded RNA. |
| Lysis Buffer (Buffer AL) | contains a chaotropic salt (guanidinium chloride) that destabilizes hydrogen bonds, van der Waals forces, proteins, and hydrophobic interactions setting up the conditions for the DNA to selectively bind to the silica resin membrane. |
| Ethanol | The ethanol will enhance and influence the binding of nucleic acids to silica by aiding the lysis buffer in creating a more hydrophobic solution |
| Detergent | has a hydrophobic side and a hydrophilic side. Because they have both polar and nonpolar ends, detergent molecules are amphipathic is a salt and breaks up positive and negative interactions of the 3-D shape as well. |
| Proteins | have hydrophobic and hydrophilic sides, the detergent is attracted to these and forces the protein apart. |
| A protein's 3-D structure | is partially created by hydrophobic and hydrophilic interactions to itself, the detergent substitutes this self bonding with detergent-amino acid bonding. |
| Wash Buffer 1 (AW1) | The AW1 wash has a low amount of chaotropic salt that binds to and removes the proteins and colored contaminants |
| Wash Buffer 2 (AW2) | The AW2 contains ethanol to remove the salts added from AW1. |
| DNA grade water | The DNA grade water free of salts, Dnases, and Proteases which allows for the re-hydration and renaturing of the DNA, causing it to lose affinity for the silica. (pH 5.4-7 at 25° C) |
| Resuspension Buffer (Buffer ATL) | The phospholipids in the membrane are also solubilized by the detergent. SDS helps release the DNA binding proteins by denaturing them and binding both membrane and non-membrane proteins as monomers. There may be an appearance of small soap bubbles. |
| Proteinase K | The addition of it degrades these nucleases and protects the nucleic acids from nuclease attack. In addition, it is stable over a wide pH range and is well suited for use in DNA extraction. |
| Rnase A | DNA will remain intact because it does not have the 2’-hydroxyl group required by it for forming the cyclic intermediate cleavage product. |
| Plasmid | small circular DNA used to move DNA from one organism into a second; vector |
| Ligation | enzymatic reaction that will “paste” two pieces of DNA together. Pieces can be from the same organisms or two different sources |
| Recombinant DNA | Term for what is produced when DNA from two different sources (chDNA + Plasmid DNA) |
| Shotgun Cloning | is the practice of randomly digesting a large piece of DNA to smaller pieces that can then be ligated into plasmids for transport to other organisms. |
| Genomic Library | collection of cloned DNA fragments from a genome.each part the genome is represented. The library can be screened for the presence of a sequence of interest. In our project the genome is from A. fischeri and the sequence of interest is the lux operon |
| Clone | A bacteria housing a recombinant plasmid that contains a piece of genomic DNA; it doesn’t have to contain the gene you are looking for. |
| Resuspension Buffer (Buffer ATL) | contains sodium dodecyl sulphate (SDS) a strong anionic detergent that can solubilize the membrane proteins. The detergent disrupts the lipid bilayer of gram-negative cells and brings the proteins into solution as protein-lipid-detergent complexes |
| Proteinase K | an enzyme that will aid in the release of nucleic acids while deactivating nucleases (enzymes that degrade nucleic acids) present. |
| RNAse | RNase A is an efficacious ribonuclease that is used to degrade RNA that is present in the sample. It cleaves the 3’ side of phosphodiester bonds after pyrimidine nucleotides in single stranded RNA. |
| Lysis Buffer (Buffer AL) | contains a chaotropic salt (guanidinium chloride) that destabilizes hydrogen bonds, van der Waals forces, proteins, and hydrophobic interactions setting up the conditions for the DNA to selectively bind to the silica resin membrane. |
| Ethanol | The ethanol will enhance and influence the binding of nucleic acids to silica by aiding the lysis buffer in creating a more hydrophobic solution. |
Created by:
tinaarnholt
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