RecombinantDNATest1

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victimsofadown
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RecombinantDNATest1
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2014-10-15 15:34:18
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RecombinantDNATest1
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  1. What is NEBcutter? What is it used for and what information does it give you?
    • Online tool where nucleotide sequences can be entered
    • Used to determine restriction enzyme cutting sites (0, 1, 2, etc)
    • Used to determine open reading frames (ORF)
    • *NOTE- ncbi ORF finder can do 6 frames easily
  2. What is BLASTn? What is it used for and what information does it give you?
    Online tool that allows you to compare 2+ nucleotide sequences (genes) and look for mutations/differences
  3. Using online tools and two sequences how can you determine if mutation will result in new AA?
    • Use NEB cutter to determine ORF
    • Use BLASTn to align the two sequences
    • Determine the nucleotide differences
    • 1) Are the mutations located within the ORF?
    • 2) If so, what position in the codon are they located? (use ORF start nucleotide as #1)
    • 3) Look up nucleotide table online and determine if AA is altered
  4. How do you determine the melting temperature of a primer?
    • A/T = 2C
    • C/G = 4C 
    • *due to stronger bond between G/C
  5. Using proper terminology explain why Primer #4 in the Cornyebacterium glutamicum experiment included the reverse complement of the end of our gene
    • For primers to properly align they must be on two different strands of DNA
    • If the end of our gene is assumed to be on the 5'-3' strand of DNA then we require the "exact opposite" to be on the 3'-5' strand
  6. Smallest increment that each pipette (20, 200, 1000) can be adjusted to?
    • P-20: 1/100 of uL (red digit shows 1/10uL)
    • P-200: 1/10 of uL (red digit shows 1uL)
    • P-1000: 1uL (red digit shows 10uL)
  7. What is pAMP? pKAN
    • pAMP: Plasmid that grants resistance to ampicillin
    • pKAN: grants kanamycin resistance
  8. Why is it important to use buffer (not water) when running agarose gel?
    • Maintain pH and temperature (prevent degradation of sample)
    • provides ions to generate current
  9. What are the functions of loading dye?
    • Provides color for sample visualization
    • Adds weight to sample ensuring it will sink to bottom the well
    • Negative charge allows DNA migration to represent bp size
  10. What happens if gel is run too long? Too short? Reverse electrodes?
    • Too long: sample will migrate past the end of the gel
    • Too short: sample will barely move and will not separate
    • Reverse: Current runs (-) to (+) and a reverse to this current will cause the sample to run backward and quickly run off
  11. What stains have we used for gels? Describe them
    • Ethidium bromide: requires destain (sodium buffer)
    • absorbs UV and produces pink visible light
    • SYBR Safe: less harmful than Ethidium Bromide
    • does not require destain process
  12. What is important to remember when determining expected band sizes of lambda DNA after an RE digest?
    • Lambda DNA infects E. coli as a linear piece w/ 2 sticky ends
    • These ends are prone to attachment and will create an "unexpected" piece 
    • size of unexpected piece = size of first piece+ size of last piece
  13. Why are bands fainter as you move from top to bottom in a gel?
    • Dye is quantitative and binds to each base pair
    • More base pairs = more dye
    • If the number of bands is distributed equally then we would expect that a 1000kpb band would be 2x as dark as a 500kbp band (2x # of bp)
  14. Describe the various Biosafety levels w/ examples
    • BSL1 - do not cause disease (E. coli)
    • BSL2 - moderate level of disease potential (Measles virus)
    • BSL3 - may cause lethal disease (HIV)
    • BSL4 - high risk of lethal disease, airborne possibility, no therapy (ebola)
  15. What are the ranges of % agarose gels?
    • 0.8% is lowest possible percent
    • 2.5% is used for smaller fragments (to hinder movement)
  16. Why use a DNA ladder when running a gel?
    • Use as a size standard (a ruler) to approximate unknowns and verify knowns
    • Serves as positive control for gel
  17. DILUTIONS - mM vs % vs X (describe each system and give examples)
    • mM: mmol/L
    • %: g/100mL 
    • X: concentration compared to working concentration (working concentration is 1X)
    • eg- 10X needs to be diluted 10 times to be working solution
  18. What is the trick to performing an X dilution where you are adding to an already existing sample?
    • You divide the existing solution amount by X-1
    • eg- if adding 6X into 30uL soln, you divide 30ul by 5 and determine that you must add 6uL of 6X
  19. Give an overview of PCR w/ proper names and estimated temperatures.  What is unique about the initial cycles?
    • Consists of a set amount of cycles, and in each cycle 3 phases occur
    • Denaturation: 95C 
    • time must ensure full denaturation
    • Annealing: 55-65C
    • allows primer to bind to exposed strands
    • Extension: 72C
    • DNA sequence is copied using primer as startpoint
  20. How do you determine how much agarose and SB to add to a gel? (Y% gel)
    • Y% gel = Yg Agarose/100mL SB 1X * 100%
    • eg- 0.8% gel = .4g Agarose/50mL SB 1X
  21. Describe the making and running of a .8% agarose gel
    • Weigh .4g agarose
    • Mix with 50mL SB  (.4g/50mL*100% = .8%)
    • Heat to dissolve
    • Assemble gel tray and comb (2mm above base) near negative pole
    • Pour gel after it reaches temperature "shock a child, but not burn them"
    • Allow gel to solidify without bumping
    • Remove comb (better late than early)
    • Overlay gel and basins with SB buffer
    • Load one DNA sample into each well (micropipetter)
    • Connect to low voltage power supply
    • *NOTE- if time must be less than 6 hours (diffusion), voltage must be less than 150 (heat)
    • add DNA stain (or have previously included) to visualize under UV light
  22. What is "SB"?
    • Sodium Borate (basically saltwater)
    • Used as a buffer for gels (0.005M in this class)
  23. What is agarose? Where does it come from?  What is it used for?  How does it work in gel?
    • Long chain sugar (highly purified form of agar)
    • Produced by a specific seaweed
    • Used to give thicker consistency to food
    • During heating the tight chains unravel and will not repack as tightly after cooling resulting in gel-like consistency
  24. What are nicked circle DNA,  multimers, and supercoiled plasmids?  How do they affect running of gel?
    • Nicked circle: broken circular DNA, larger surface area causes less movement down the gel
    • Multimers: when crossing over occurs between plasmids the plasmids join
    • if multimers are run on a gel the larger surface area will cause less movement on the gel
    • Supercoiled: highly condensed DNA (plasmid)
    • moves further down the gel than would be expected
  25. How can ladders sometimes be misleading?
    • Circular and linear DNA cannot be compared on a gel
    • If the ladder is not the same type of DNA being observed then the ladder will be completely irrelevant
  26. What is the ideal temperature for DNA ligase?
    • enzyme activity increases with temperature, but so does the movement of particles
    • Since DNA ligase is attempting to catalzyse the bonding of sticky ends we have to find a balance between efficiency of enzyme and fleeting interactions of sticky ends
    • We attempted 5h at 16C, 5h at 24C, then overnight at 20C
  27. What is the NSABB?
    • National Science Advisory Board for Biosecurity
    • US organization that advises scientific community on policies re public disclosure
  28. define transfection, transformation
    • transfection: introducing foreign DNA into eukaryotic cells
    • transfection: "" into prokaryotic cells
    • transfection is much easier
  29. What is induced transformation and what are its variations?
    • untreaded plasmid DNA and bacteria yield very low DNA uptake
    • induced transformation results in thousands of transformed cells/ug plasmid DNA
    • heat shock: classical method
    • electroporation: electric discharge temporarily disrupts membrane allowing plasmid into cell, then quickly resealing (bacteria or plant cells most common)
  30. What is a nuclease and what are its subcategories?
    • Nucease: enzyme that cleaves DNA (DNAse) or RNA (RNAse)
    • exonuclease: cleaves from the end of nucleotide chain
    • endonuclease: cleaves from the middle
  31. What are Restriction Enzymes?  Where do they come from?
    • Naturally occurring endonucleases
    • Found in many bacteria
    • Now commercially available
  32. Describe the naming of restriction enzymes
    • letter 1 (capital): genus of bacteria
    • letters 2-3 (lowercase): species of bacteria
    • letter 4 (capital): strain of bacter
    • *NOTE- 4th letter may be absent
    • numeral after name: order of discovery from that strain
    • EG- EcoR I (the first RE found in Eschericia coli R strain)
  33. What trick can you use for finding a potential RE site in a string of nucleotides?
    • Scan the nucleotides for complementary pairs (CG  or TA on the same strand)
    • Work outward from this point and look for a reverse complement of ~6 nucleotides
    • EG- 5'-3' GACGTC and 3'-5' CTGCAG
  34. Describe the various ends that a restriction enzyme can form
    • 5' overhangs: cuts asymmetrically within site such that a short single stranded segment extends from 5' ends
    • 3' overhangs: cuts asymmetrically within site such that a short single stranged segment extends from 3' ends
    • blunt ends: no overhangs exist (no sticky ends)
  35. What is 1 unit in terms of RE sales?  Is this useful?
    • Amount of time to digest 1ug lambda DNA in 1 hour under ideal conditions
    • Not very useful, what if lambda DNA has only 1 site and your gene has 10?
  36. Why can't we save money and digest overnight with only a few units of RE?
    Risk of nuclease degradation by contaminated DNAses is too high
  37. How do you store restriction enzymes?  Why?
    • In a -20C non-frost-free freezer, suspended in 50% glycerol
    • Frost free-freezers heat up periodically to eliminate frost, that will denature the enzymes
    • Glycerol prevents freezing
    • *NOTE- if too much glycerol is present then solution is too viscous
  38. What is star activity?
    • Some RE are capable of cleaving similar, but nonidentical, sequences. (problem)
    • This is especially true under extreme conditions
  39. What is the maximum amount RE in final soln?
    10% of total volume, otherwise star activity will occur
  40. What are the various RE buffers, and what is their purpose?
    • TRIS buffer: maintains constant pH
    • MgCl2: Mg++ are RE cofactor
    • NaCl: proper osmotic conditions
    • beta-mercaptoethanol: reducing agent
    • dH2O: base for almost every buffer
  41. Describe Primer relevance and general cloning scheme of overarching project
    • Primer 1: RE site
    • Primer 2: overlaps #3
    • Primer 3: re site
    • Primer 4: includes HIS tag
    • General scheme: amplify bacterial promoter (p1,p2) and gene (p3,p4) separately
    • amplify those products together (p1,p4)
    • Insert final product into new plasmid for entry into wt Cornyebacterium glutamicum
  42. What is the purpose of PCR?
    Spcifically targets and amplifies a single sequence from a mixture of DNA
  43. What are the basic components required in PCR?
    • deoxynucleotide triphosphates (dNTPs): A, T, G, C
    • DNA polymerase: must be thermal stable (eg. Taq polymerase)
    • Primers: provide specificity to replication
    • Template DNA: plasmid DNA is usually used
    • Buffer
    • MgCl2
  44. Why is the first cycle of PCR inefficient?
    The true product doesn't appear until cycle 3 (it is only cut on one side before then)
  45. What happens if annealing temperature is too high/low in PCR?
    • too high: primers bind to non-exact locations
    • too low: primers won't bind at all
  46. What is multiplex PCR?
    PCR that can copy 10-16 regions simultaneously with sensitivities of <1ng DNA
  47. What applications has PCR improved?
    • Forensic DNA technology
    • prenatal diagnosis
    • Ancient DNA amplification for study
  48. What are pros/cons of PCR?
    • PROS: highly sensitive
    • small sample needed
    • fast results
    • cell-free
    • CONS: highly sensitive
    • contamination easy (product carryover)
    • must be extremely careful with equipment if using same primer
    • *negative controls (all amp requirements w/o DNA) are necessary
  49. EXPERIMENT SPECIFIC- Gene start RE? End RE? size of gene
    • Gene start RE: BamHI and SacI
    • Gene size: 912bp

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