Card Set Information
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?
: 1/100 of uL (red digit shows 1/10uL)
: 1/10 of uL (red digit shows 1uL)
: 1uL (red digit shows 10uL)
What is pAMP? pKAN
: Plasmid that grants resistance to ampicillin
: 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?
: sample will migrate past the end of the gel
: sample will barely move and will not separate
: 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
: requires destain (sodium buffer)
absorbs UV and produces pink visible light
: 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)
: 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
time must ensure full denaturation
allows primer to bind to exposed strands
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?
: broken circular DNA, larger surface area causes less movement down the gel
: 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
: 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
: introducing foreign DNA into eukaryotic cells
: "" 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
: classical method
: 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?
: enzyme that cleaves DNA (DNAse) or RNA (RNAse)
: cleaves from the end of nucleotide chain
: 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
: cuts asymmetrically within site such that a short single stranded segment extends from 5' ends
: cuts asymmetrically within site such that a short single stranged segment extends from 3' 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?
: maintains constant pH
: Mg++ are RE cofactor
: proper osmotic conditions
: reducing agent
: base for almost every buffer
Describe Primer relevance and general cloning scheme of overarching project
: RE site
: overlaps #3
: re site
: includes HIS tag
: 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
: must be thermal stable (eg.
: provide specificity to replication
: 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?
: primers bind to non-exact locations
: 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
Ancient DNA amplification for study
What are pros/cons of PCR?
: highly sensitive
small sample needed
: 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