1. | An enzyme that breaks a phosphodiester bond in DNA. DNA ligase creates a phosphodiester bond. | A. | compatible cohesive ends |
2. | the use of experimental techniques to produce DNA molecules containing new genes or new combinations of genes. | B. | expression vectors |
3. | Uses toposomerase instead of ligase to complete bond. Forward primer designed with CACC; vector has GTGG overhang which can invade end of insert (“strand invasion”). Directional cloning and no ligase! | C. | SDS |
4. | A type of ion-exchange chromatography that uses a column and is based on charge | D. | ddNTP |
5. | Consists of double digestion with two different enzymes that leave incompatible ends in order to prevent vector from re-ligating (after removal of stuffer fragment). Insert DNA is cut with same two enzymes used to cut vector. Another advantage is that the orientation of the insert is forced. | E. | Ponceau Red |
6. | In this course, this was the original source of the GFP | F. | Lowry protein assay |
7. | During ligation with insert, plasmid vectors have a tendency to re-ligate upon themselves (to generate original circular plasmid) rather than with an insert. Phosphatase treatment and forced cloning can minimize vector re-circularization. Remember that ligation only occurs in the presence of an enzyme such as DNA ligase. | G. | screen |
8. | are DNA ends produced by restriction enzymes that recognize different sites but create staggered ends that are complementary. Hybrid sites formed by ligation usually can't be re-cleaved by either enzyme. | H. | BCA assay |
9. | take up the bottom 2/3 to 3/4 and is where the proteins resolve into discreet bands by size. | I. | Recombinant protein |
10. | A cut left of center produces cohesive ends with 5' overhands or a cut to the right of center 3' overhangs. Staggered cuts. | J. | plasmid |
11. | Protein assay that uses a dye that binds primarily to basic (especially arginine) and aromatic amino acid residues. It is not dependent on single amino acids. However, it is not compatible with detergents and some common protein buffers. | K. | Dicer |
12. | Useful if cloning with only one enzyme and in blunt-ending, since vector re-circularization can occur.Treat only the vector with alkaline or antarctic phosphatase (newer). AP removes 5' phosphates from ends of DNA. Vector cannot re-ligate upon itself; it can only ligate to insert. | L. | gene cloning |
13. | reducing agent, breaks up covalent cysteine (disulfide) bonds | M. | Coomassie blue |
14. | This is the classic method, but is rarely used. A major disadvantage is that it is dependent on tyrosine and tryptophan residues. It is not compatible with detergents and not ideal for purified proteins. | N. | sticky or cohesive ends |
15. | polypeptide that is synthesized in a recombinant cell as the result of expression of a cloned gene. | O. | Bradford protein assay |
16. | is added to DNA sample before loading the gel. It contains glycerol which is heavier than TBE or TAE buffers and allows sample to sink. It also contains one or more dyes (e.g., bromophenol blue) that serve as tracking dyes. Also called "DNA loading dye" or "sample buffer". Usually made as a 10X solution. | P. | Gateway cloning |
17. | are primarily used to make recombinant protein. Expression vectors have everything necessary to insert foreign DNA and to replicate, and also contain everything necessary for gene transcription and translation. Often have encoded tags used for protein purification. | Q. | TA cloning |
18. | Recognition sequence on DNA that restriction enzyme binds to and cleaves the phosphodiester bond. Usually palindromic (read the same 5' to 3' on both strands), but the sites can vary both in nucleotide sequence and length. Different enzymes recognize distinct sites. | R. | Bromophenol blue |
19. | A mini column with a silica membrane. Plasmid DNA selectively adsorbs to membrane. Plasmid DNA binds membrane under high salt conditions. Impurities are washed away. Pure plasmid DNA is eluted under low salt conditions. Silica columns are used in "mini prep kits" | S. | cloning vectors |
20. | tracking dye that is small enough to migrate faster than most proteins | T. | ligate |
21. | is a catalytic subunit of RISC – Binds siRNA and cleaves the passenger strand | U. | restriction endonuclease or restriction enzyme |
22. | is acatalytic subunit of RISC – Binds siRNA and cleaves thepassenger strand | V. | anion exchange chromatography |
23. | are primarily used to propagate DNA and have everything necessary to insert foreign DNA and to replicate, but don't usually transcribe the inserted DNA. Cloning vectors sometimes have screenable markers that allow for the identification of positive clones. | W. | blunt ends |
24. | Clone genes in frame into a Gateway donor vector/entry clone and easily move them into multiple different expression vectors using homologous recombination (flanking att sites from phage). | X. | DNA loading buffer |
25. | DNA created in vitro by joining together pieces of DNA that are not normally contiguous. | Y. | affinity resin |
26. | Uses tethered topoisomerases (on vector) instead of ligase to complete the bond. Sticky ends from T on vector and A on insert. No ligase needed, but not directional | Z. | vector |
27. | The affinity resin used for affinity chromatography and protein purification | A1. | restriction sites |
28. | Plasmid purification process that enables the separation of plasmid DNA from chromosomal DNA | B1. | Argonaute (Ago) |
29. | HRP colorimetric substrate. Used with goat anti-mouse antibody conjugated to horseradish peroxidase (GAMP, our secondary antibody) to detect positive signals. | C1. | glycerol |
30. | Coomassie binds specific amino acids, after destaining , blue bands representing each protein | D1. | Forced or directional cloning |
31. | a protein made by splicing two genes or gene sequences together which, when translated,produce a hybrid or chimeric protein (e.g., GST::EGFP). In our case, the tag is fused to EGFP to simplify protein purification. Depending on the vector used, the tag can be cleaved off using a protease. | E1. | Beta mercaptoethanol |
32. | the use of living organisms for industrial processes | F1. | Humulin |
33. | Heat-labile antibody against Taq polymerase in PCR mix to reduce nonspecific amplification of products from low-temperature priming. Keeps the polymerase inactive until cycles begin in thermocycler. | G1. | RNase H |
34. | insertion of a fragment of DNA, carrying a gene, into a plasmid vector, and the subsequent propagation of the recombinant DNA molecule in a host organism. Subcloning is when cloned DNA is moved from one plasmid to another. | H1. | RISC |
35. | Top 1/4 to 1/3 of gel. Proteins form a "stack" where proteins of all sizes line up in one band so they can all start migrating from the same point.The stacking gel has a nonrestrictive large-pore size (less acrylamide). | I1. | select |
36. | Specific ligand for glutatathione-S-transferase (GST). Used in BIT 410/510 to purify your GST::EGFP fusion protein. | J1. | TOPO TA cloning |
37. | Ponceau Red is temporary/ reversible used to verify if transfer was successful | K1. | Stacking gel |
38. | A cut at the center of dyad = blunt ends | L1. | Hot start PCR |
39. | An enzyme that creates a phosphodiester bond. Requires ATP.DNA ligase catalyzes covalent bond formation between 3’OH and 5’PO4 on DNA (phosphodiester bond). | M1. | Chloronaphthol |
40. | Use Taq polymerase to add a single-non templated nucleotide (nearly always A) to the 3’ends of DNA. Use it as a sticky end in conjunction with a “T-vector”. Not directional. | N1. | Running or resolving gel |
41. | assay that is similar to Lowry but has the advantage of being compatible with detergents. . | O1. | recombinant DNA |
42. | to introduce recombinant molecules into a prokaryotic or eukaryotic cell to serve as a host for DNA amplification. | P1. | phosphatase treatment |
43. | Used in loading dyes (or protein sample buffer): facilitates the sample sinking to the bottom of the well. | Q1. | Alkaline lysis |
44. | Gel Red binds specifically to DNA and is excited by UV light; therefore when the gel is exposed to UV light you can visualize the DNA. Gel Red is used in the lab instead of ethidium bromide. | R1. | Argonaute (Ago) |
45. | the first recombinant DNA drug approved by the FDA in 1982. Scientists at Genentech cloned the gene for human insulin. Genentech licensed the technology to Eli Lilly. | S1. | DNA ligase |
46. | a circular piece of DNA, independent of the host chromosome, capable of replication. | T1. | insert |
47. | RNase H digests RNA from RNA/DNA hybrids. It is used when making cDNA from mRNA. | U1. | Aequorea victoria Jellyfish |
48. | DNA of interest to be cloned | V1. | Biotechnology |
49. | to covalently join (paste) different pieces of DNA, usually a vector and insert. | W1. | Champion pET directional TOPO cloning |
50. | to identify cells that contain recombinant DNA of interest (clones) | X1. | Gel Red |
51. | to identify cells that received a plasmid using a selectable marker such as an antibiotic or nutritional requirement. | Y1. | Genetic engineering / molecular biology |
52. | a DNA molecule (often a plasmid) capable of replication in a host organism, specially modified for propagating DNA sequences. | Z1. | transform |
53. | The special nucleotide (dideoxynucleoside triphosphate, ddNTP) is a chain terminator because it has a H instead of OH in the 3’ position so there is no “end” to use in order to form a phosphodiester bond and strand elongation is halted. | A2. | Glutathione |
54. | Dicer is an ATP-dependent RNaseIII -like protein which progressively cleaves the long dsRNA into small dsRNAs | B2. | fusion protein |
55. | Dicer and a binding partner protein (BP) load the siRNAs into a RNA- induced Silencing Complex (RISC). RISC processes the siRNAs so that they will be able to interact with complementary mRNA | C2. | vector re-circularization |
56. | anionic detergent that denatures proteins by wrapping around the polypeptide backbone(forming a micelle), and thus confers a net negative charge in proportion to polypeptide length. SDS also disrupts most non-covalent bonds, decreasing protein folding. | D2. | silica adsorption |