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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
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
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
How do you determine the melting temperature of a primer?
- A/T = 2C
- C/G = 4C
- *due to stronger bond between G/C
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
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)
What is pAMP? pKAN
- pAMP: Plasmid that grants resistance to ampicillin
- pKAN: grants kanamycin resistance
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
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
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
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
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
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)
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)
What are the ranges of % agarose gels?
- 0.8% is lowest possible percent
- 2.5% is used for smaller fragments (to hinder movement)
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
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
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
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
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
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
What is "SB"?
- Sodium Borate (basically saltwater)
- Used as a buffer for gels (0.005M in this class)
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
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
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
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
What is the NSABB?
- National Science Advisory Board for Biosecurity
- US organization that advises scientific community on policies re public disclosure
define transfection, transformation
- transfection: introducing foreign DNA into eukaryotic cells
- transfection: "" into prokaryotic cells
- transfection is much easier
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)
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
What are Restriction Enzymes? Where do they come from?
- Naturally occurring endonucleases
- Found in many bacteria
- Now commercially available
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)
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
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)
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?
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
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
What is star activity?
- Some RE are capable of cleaving similar, but nonidentical, sequences. (problem)
- This is especially true under extreme conditions
What is the maximum amount RE in final soln?
10% of total volume, otherwise star activity will occur
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
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
What is the purpose of PCR?
Spcifically targets and amplifies a single sequence from a mixture of DNA
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
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)
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
What is multiplex PCR?
PCR that can copy 10-16 regions simultaneously with sensitivities of <1ng DNA
What applications has PCR improved?
- Forensic DNA technology
- prenatal diagnosis
- Ancient DNA amplification for study
What are pros/cons of PCR?
- PROS: highly sensitive
- small sample needed
- fast results
- 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
EXPERIMENT SPECIFIC- Gene start RE? End RE? size of gene
- Gene start RE: BamHI and SacI
- Gene size: 912bp
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