CTE Skills Test Review

1. CTE Skills Test Review

2. LAB ATTIRE • Closed toe shoes • Lab coat • Goggles • Gloves • Hair tied back • No cosmetics • No gum, food, or drink

3. DISPOSAL • Glass • Glass waste disposal box • Bacteria • Bleach • Autoclave • Other waste • Proper garbage disposal

4. LAB NOTEBOOK • Date • Initials • Number each page • Table of contents

5. LABELING • Reagent bottles, bacterial plates, etc. • Initials • Date • What is in and/or on • Concentration (M or %) • On the bottom of the plate [part with agar]

6. ASEPTIC TECHNIQUE • Disinfect counters and work surfaces. • Flaming loop, spreader, etc.

7. DISINFECTION • Always clean counter before and after • Use • 70% ethanol • Bleach • Other disinfectants

8. EFFECT OF UV ON BACTERIA • Kills bacteria if you leave it under UV light

9. AUTOCLAVE • Uses high heat and pressure to sterilize objects

10. MICROPIPETTES • 1000 ul = 1 ml • Be within the range but not on the end with amounts to pipette • P20, p100, p200, p1000

11. CHEMISTRY EQUATIONS • Molarity (M) = moles/liter • moles = grams/molar mass • Molar mass is the sum of the atomic mass of elements • M = [grams/molar mass]/liter • Volume % = volume solute/volume solution x 100 • M1V1 = M2V2 • C1V1 = C2V2

12. BONDS • Hydrogen- hydrogen bond with other element • Water, DNA between nitrogen bases, proteins • Covalent- sharing of a pair of electrons by two atoms • DNA on backbone • Ionic- through transfer of 1+ electrons • ions • Buffers- salt, etc. • Disulfide- binds peptide chains • Gives protein 3D shapes [usually between chains] • Peptide- bond amino acids, remove a molecule of water (dehydration) • Amino acid bonds

13. HYDROPHOPIC/HYDROPHILIC • Part of the membrane • Hydrophobic • Water fearing • Tails • Hydrophilic • Water loving • Head

14. POLAR/NON-POLAR • Polar • Has a charge • +/- • Ex: water • Non-polar • Doesn’t have a charge • Ex: sugars, oils

15. pH • Salt concentration can change the shape of proteins • Change in acid and bases can kill enzymes • Acids • Lower pH to 1>7 • Bases • Raise pH to 7<14

16. STOCK SOLUTIONS • Dilute stock solution to get desired solution concentration • C1V1 = C2V2

17. SPECTROPHOTOMETRY • An instrument used to determine the intensity of various wavelengths in a spectrum of light • Can determine concentrations of solutions • Can make a graph of OD and absorbance v. concentration • Can change the wavelength to find the protein, DNA, RNA, and bacterial concentration

18. SERIAL DILUTION • Dilute to get smaller and smaller concentrations • Can go from lawns to single colonies, an alternative to streaking

19. Prokaryotic cells No nucleus Eubacteria Archaebacteria- extreme bacteria Operon (grouped genes) Don’t have introns Eukaryotic cells Nucleus Protists Fungi Plants Animals DNA has introns and exons (splice introns out and bind exons together to form mRNA) PROKARYOTIC V. EUKARYOTIC

20. MEDIA PREPARATION & INCUBATION • Media preparation- can manipulate what you grow • Incubation • 37°C if grown inside body • 25°C if grown elsewhere • LB/AMP/ARA • LB- nutrients necessary for bacterial growth • AMP- ampicillin resistance to see if there was an uptake of the protein • Selective procedure- only ampicillin resistant bacteria can grow • ARA- arabinose to turn on the GFP • Allows the gene to be transcribed (operon involved)

21. ANTIBIOTIC RESISTANCE FOR SELECTION • Antibiotic- natural substance secreted by 1 microorganism that will kill or inhibit growth and reproduction in other microorganisms • Shows if there was an uptake of the new genes

22. INOCULATION OF MEDIA • Streaking • Proper lab attire…not that kind of streaking! • Get a single colony on you loop and streak it across the plate in a zig-zag fashion. Turn it a quarter turn, flame and repeat. • Spreading • Pipette LB broth onto plate and sterilize paperclip. Spread broth with paperclip over the entire gel.

23. IDENTIFYING MICROORGANISMS • Colony Morphology • Size • Shape • Margin • Elevation • Texture • Light transmission • Color • Antibiotic resistance • Incubation temperature • Gram staining

24. BACTERIA TYPES • Cocci • round • Bacilli • rod • Spiral • Staph- • Grape-like clusters • Strep- • Chains • Diplo- • Two together

25. GRAM STAINING • Depends on the structure of the cell wall • Gram positive • Purple • Gram negative • Red

26. DNA STRUCTURE • Runs 5’ to 3’ • Sugar (deoxyribose) • Phosphate (phosphoric acid) • Negative charge (allows for electrophoresis) • Nitrogen bases • 2 hydrogen bonds • Adenine – Thymine • Gene cutting happens most often here • 3 hydrogen bonds • Guanine – Cytosine

27. DNA REPLICATION ENZYMES • Helicase • Splits the DNA molecules apart • RNA primase • binds primers (RNA nucleotides) by complimentary base pairing • DNA polymerase • Adds nucleotides to extend the DNA • Binds leading DNA strand starting at 3’ end • TAQ polymerase can withstand high heat • RNA primers are removed • Ligase • Seals gaps in the sugar phosphate backbone

28. RNA • Ribose sugar • has one more oxygen molecule • Phosphate • Nitrogen bases • Adenine – URACIL • not thymine • Guanine – Cytosine

29. TRANSCRIPTION • DNA to RNA • Eukaryotes • Eukaryotes have introns and exons; the introns are removed • 5’ cap and 3’ poly-A tail on the exons that have been spliced together • Prokaryotes • Operons

30. PROTEINS • Peptide bonds • Eukaryotic • Multiple proteins from 1 RNA • Prokaryotic • Operon

31. PROTEIN STRUCTURE • Primary • Amino acid sequences • Secondary • Alpha-helices or beta-sheets • Tertiary • Domains • Quaternary • Subunits

32. FUNCTIONS OF PROTEINS- REST! • Regulatory • Genes and cellular processes are turned on and off • Enzymes • All enzymes are proteins but not all proteins are enzymes • Covalent bond breakage and formation • Structure • Mechanical support to cells and tissues • Transport • Move things in and out of the cell

33. PROTEIN SYNTHESIS • RNA to protein • Initiation • Elongation • Termination • The stop codon doesn’t code for an amino acid • mRNA • coded DNA • Codons • tRNA • transfers amino acid • Anti-codons • Amino acids

34. AMINO ACID CHARACTERISTICS • Peptide bonds (dehydration) • Water • Hydrophilic • Hydrophobic • Charge • Positive • Negative • Polarity • Polar • Non-polar • Uncharged polar

35. DNA FINGERPRINTING • Identifies matching DNA fragments (bands) • RLFP- Restriction length fragment polymorphisms • VNTR- Variable Nucleotide tandem repeats • Introns- non-coding sequence that varies

36. RESTRICTION ENZYMES • Also called endonucleases • Specific sequences of DNA nucleotides • Cut at specific places • Can be palindromes (same forwards and backwards) • Come from bacteria • To cut up viral DNA • Methylate own DNA to protect it

37. RESTRICTION DIGEST • Where you cut DNA with restriction enzymes • Results in DNA fragments • Can then be run on gel electrophoresis

38. GEL ELECTROPHORESIS • Gel electrophoresis- uses electric current to separate DNA fragments by size • Runs to red (positive) • Bands • Various sized fragments of DNA • Introns • Pieces • Smallest ones run the farthest

39. SDS-PAGE ANALYSIS • Run vertically • Mostly used for protein • Smaller pores than agarose • Smaller matrix • Sorts by size and charge

40. PCR • Polymerase Chain Reaction • Denature • Raise the temperature to unzip DNA • Anneal • attach primers • Extend • Binds TAQ polymerase • TAQ polymerase can withstand high heat • Heat and cool • 1 million copies for 30 cycles

41. DNA SEQUENCING • Used to know the nucleotide sequence of the human genome • Process • Put DNA with DNTPs • Terminates elongation of DNA • PCR • Run on a gel to tell the sequence of the nucleotides

42. READING FRAME • Frame shift mutations • Point mutations • Deletion • Insertion

43. RECOMBINANT DNA • 2 different pieces of DNA combined • Use restriction enzymes to cut the gene of interest and the plasmid • Insert the gene of interest into the plasmid

44. TRANSFORMATION • Insertion of a gene into bacteria • Competent cells- take up the plasmid • Restriction enzymes- cut the plasmid • Selection- so you can get the colonies with the selected gene • Antibiotic resistance

45. PLASMID • Origin of replication • Allows plasmid to self-replicate • Antibiotic resistance • Allows for the selection of the desired bacteria • Operon • Turns on the gene of interest • Gene of interest • Example: GFP