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179 Cards in this Set
- Front
- Back
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Fluorescence can be used to quantify DNA. TRUE OR FALSE
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TRUE |
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List different types of fluorescence |
Ethidium Bromide (EtBr) - most common which binds to dsDNA Others: PicoGreen, SYBR Green I, Hoechst - more sensitive but more expensive |
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What does OD280 measure? |
Measures aromatic amino acids (protein contamination) |
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What does OD230 measure? |
Measures contaminating organics and proteins (primarily organics) |
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OD260/280 and OD260/230 measure what? |
purity |
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For pure DNA, OD260/280 and OD260/230 are greater than or equal to _______. |
1.8 |
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For pure RNA, OD260/280 AND OD260/230 are greater than or equal to _______. |
2.0 |
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For concentration of DNA (ng/microliter), it should not be more than..? |
2000; if it is, it needs to be diluted otherwise there will be inconsistency |
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How does a Nanodrop work? |
Surface tension is used to hold a column of liquid sample between the ends of two optical fibers to establish a measurement path. A beam of light is shown through the water column and is measured |
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What does Protease K do? |
Enzyme that breaks up proteins |
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What does guanidinium thiocyanate do? |
Very strong detergent that breaks up proteins in cells |
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Why do we flush DNA with water? |
To break the ionic bonds |
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What is the purpose of an RNA primer and how does it get to the DNA strand? |
RNA primase adds an RNA primer to the 3' end of the parent strand. DNA polymerase binds to the primer and adds nucleotides complementary to parent strand in the 5' to 3' direction. After synthesis of the daughter strand, DNA polymerase removes the RNA primer. |
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What does DNA ligase do? |
forms the new phosphodiester bonds to link the okazaki fragments |
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DNA polymerase synthesizes in the 3' to 5' direction. TRUE OR FALSE |
FALSE; 5' to 3' direction |
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Leading strands v lagging strand |
continuous replication v fragments replication |
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What does it mean to say 3' to 5'? |
We are referring to two different Carbon attachments on the sugars |
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What does DNA helicase do? |
Unzips the DNA |
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Pyramidines are which two bases? |
C and T |
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Purines are which two bases? |
A and G |
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Nucleotides are separated into what two groups? |
Purines and Pyramidines |
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What is the DNA backbone made up? |
Repeating phosphates and sugars linked by phosphodiester bonds |
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Which pair of bases are linked by 3 hydrogen bonds vs 2? |
A and T are linked by 2 hydrogen bonds vs C and G which are linked by 3 |
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Differences between DNA and RNA |
DNA: deoxyribose sugar, double stranded, one hydroxyl group, Thyamine RNA: ribose sugar, single stranded, two hydroxyl groups, and Uranine |
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What does DNA wrap around before forming a chromosome? |
Histones |
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Where is DNA found? |
In the nucleus of almost all cells |
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_________________ are the building blocks of DNA and help it to form into a signature double helix. |
Nucleotides |
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What two things are needed to start the DNA cloning process? |
Plasmid and foreign DNA |
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More mRNA = ? |
more protein |
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What is the central dogma? |
DNA (replication through DNA polymerase) to RNA (transcription through mRNA) to Protein (translation by ribosomes) |
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Describe forward pipetting |
First stop... up... second stop... up |
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Describe reverse pipetting |
Second stop... up... first stop... second stop... up |
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Describe repetitive forward pipetting |
First stop... up... second stop... up... (next round) first stop... up... second stop... up... |
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Describe repetitive reverse pipetting |
Second stop... up... first stop... up... (next round) first stop.. up.. |
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List generic applications of biotechnology |
Agriculture, Medicine, Industrial use, environmental, genetics and cloning |
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What is biotechnology? |
Any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use |
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What is the modern method to isolate genomic DNA? |
1) Lysis of cells or tissues (with guanidinium thiocynate) 2) Addition of silica-based beads or filters 3) DNA elution with water |
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What do the beads help with when isolated DNA? |
Help to disrupt the sample |
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What is the main enzyme responsible for DNA replication?
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DNA polymerase |
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What are the three steps of PCR? |
Denaturation Annealing Extension |
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What does PCR stand for? |
Polymerase Chain Reaction |
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About how many cycles do we do in PCR?
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25 to 35 cycles |
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What does complete separation allow for during denaturation? |
Allows the primers to bind |
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What is the reaction time and temp during denaturation? Why is this important? |
94 degrees celsius for 1 minute - Required to completely break the H bonds between bases |
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Purpose of denaturation: |
Responsible for dissociating the double stranded DNA to form 2 individual strands |
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What are the two types of matrices that could be used for electrophoresis? Which is most common? |
Polyacrylamide and Agarose gel - AGE is most common |
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Why is AGE most common? |
Cheap and easy |
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The higher the agarose concentration, the ____________ the pores and the _________ the DNA migrates.
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smaller; slower |
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What does TAE stand for? |
Tris base = acetic acid = EDTA, pH 8.0 |
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Which is more common, TAE or TBE? |
TAE |
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TBE has lower buffering capacity to keep pH where it needs to be. TRUE OR FALSE |
FALSE; TAE does |
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Smaller DNA will migrate faster. Why? |
Larger DNA will take longer to wind through the pores |
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List advantages to TAE (4) |
1. Lowering capacity 2. DNA migrates slightly faster 3. Less resolution for small DNA fragments (<500bp) 4. Less expensive |
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The electrophoresis has a positive charge on top and a negative charge on bottom. TRUE OR FALSE |
FALSE; the negative charge is on top and the positive charge is on bottom because DNA is negatively charged and opposite charges attract to pull the DNA down |
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Define chelate |
to bind |
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Why is it important to keep the pH at 8.0? |
To keep the PO4 group of DNA deprotonated and maintain its negative charge |
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What is EDTA used for? |
To chelate divalent cations and prevent DNA from being degraded by DNases or other DNA modifying enzymes |
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Why are dyes used in electrophoresis? |
Increase density of the samples Simplify the loading process with color dyes Keep track of DNA migration |
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If the DNA is super long, what color dye should you focus on? |
Blue |
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If the DNA is super small, what color dye should you focus on? |
Orange because the DNA will migrate fast |
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What does glycerol do in relation to AGE? |
Helps the DNA end up at the bottom of the wells and not float in the tankk |
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How is the separation of samples viewed in AGE?
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By UV light |
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How does EtBr bind to DNA and why is EtBr added to the agarose mixture? |
Binds to duplex DNA by intercalations with no sequence preference; has a 20 to 30 fold fluorescence yield |
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How does SYBR green bind to DNA and why? |
Binds to the minor grooves of DNA; fluorescence yield is 100 fold |
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Why is SYBR green not used more than EtBr? |
Needs more DNA and is more expensive |
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List 3 factors that effect migration rate of DNA our of the 6 |
1. Molecule size of DNA 2. Concentration of agarose 3. Conformation of DNA (superhelical circular > linear > nicked circular) 4. Applied voltage (higher voltage = faster migration) 5. Composition of the electrophoresis buffer 6. Presence of EtBr |
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Recovery of DNA from agarose gels is possible. TRUE OR FALSE |
TRUE |
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Why would you need to recover DNA from agarose gels? |
Subsequent restriction enzyme digestion, ligation to a vector for bacterial transformation, transfection, PCR and DNA sequencing |
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How is DNA placed into a plasmid? |
Using DNA ligase |
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What is the goal of PCR product? |
To have unlimited supply |
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What are restriction enzymes? |
Endonucleases that cut DNA at precise locations |
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What is a palindrome? |
Reads the same word forward and backward |
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What is a blunt cut? an overhang? |
Can end up with two different cuts: blunt or overhang blunt is even, overhang has extras at end up cut |
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Describe the nomenclature of restriction enzymes
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1. first letter = GENUS 2. next two letters = species name 3. Next letter = STRAIN 4. Number (roman) = order of enzyme discovery in each organism |
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Sticky ends are good with DNA cutting patterns of REs. TRUE OR FALSE
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TRUE |
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260/280 is measuring what? |
protein contamination |
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260/230 is measuring what? |
organic contamination |
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1 OD260 = ______ ug/ml for ds DNA |
50 |
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1 OD260 = _____ ug/ml for RNA |
40 |
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1 OD260 = _______ ug/ml for ss DNA |
33 |
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1 OD260 = ______ ug/ml for ss oligonucleotides |
20 |
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For pure DNA, OD260/OD280 and OD260/OD230 should be > _____. |
1.8 |
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For pure RNA, OD260/OD280 and OD260/OD230 should be >_______. |
2.0 |
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What is the reaction time and temp for annealing? |
54 degrees C for 45 seconds |
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Why do we decrease temperature from 94 to 54 during the annealing process of PCR? |
To allow the primers to bind |
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How is the exact time and temp deermined in the annealing process? |
G-C content and melting point; a lot of G-Cs means temp needs to increase in order to break all the H bonds because these pairs have 3 H bonds in comparison to A-T which only have 2 H bonds |
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What happens during the annealing process? |
Primers are added to allow DNA polymerase to form the new strand |
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Why do we use forward and reverse primers in annealing? |
To increase efficiency and allow for exponential amplification: need two primers to bind to the two different strands |
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What is the time and temp during extension? |
72 degrees C for 2 min |
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How is the reaction time and temp determined in extension? |
Determined to allow the DNA polymerase to work optimally |
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What are dNTPs? |
Include all four bases which is included in the solution |
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What is triphosphate used for in dNTPs? |
Provides the energy to form the phosphodiester bonds |
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how is the number of cycles determined in PCR? |
By the basal expression of genes and how much template of DNA was used |
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What is the temp and time for initial denaturation? |
94-95 degrees C for 0.5 to 5 min |
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What is the temp of and time for final extension? |
68 to 72 degrees C for 2-5 min |
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What is the purpose of final extension in PCR? |
Making sure all DNA is copied completely |
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List the PCR components |
Template Primer DNA polymerase dNTPs Divalent cations pH Ionic strength |
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What three parts of the PCR components make up the Buffer? |
Divalent cations Ionic strength pH |
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Why do you want Y as close to 1 as possible in the amplification theory? |
Because of nonspecific amplification |
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What is the PCR amplification theory? |
Nf = N0 (1 + Y)^n Nf = final copy # of target sequence N0 = initial copy # Y = amplification efficiency per cycle n = # of PCR cycles |
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What are limiting factors that lead to the plateau phase of the PCR amplification theory? |
1. Exhaust dNTPs 2. Exhaust primers 3. Enzymatic activity of Taq goes down 4. pH leaves the 8.0 to 9.0 optimal range |
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What three things do you need for PCR Optimization? |
Higher efficiency Higher specificity Higher fidelity |
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List ways to improve PCR optimization for efficiency and specificity (5) |
1. PCR program optimization: annealing temp; touchdown 2. Primer design 3. Hot start DNA polymerase: platinum Taq 4. Template clean up 5. Buffer conditions |
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What temp does Taq work optimally at? |
72 degrees C but still works at lower temps slower |
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What is platinum taq? |
Polymerase + antibody |
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More template leads to ______ amplification cycles. |
less |
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What are primer dimers? |
When two primers bind to each other and never touch PCR |
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How can you avoid primer dimers? |
Primer design and/or the annealing temp |
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What is the purpose of DNase? |
To degredate DNA |
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What enzyme performs reverse transcription?
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Reverse transcriptase |
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What is the goal of a PCR product? |
To have unlimited supply |
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DNA is placed into a plasmid using _________________________. |
DNA ligase |
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When the circular plasmid is placed inside bacteria, how quickly does it double? |
Every 20 min. |
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What is recombinant DNA? |
Cleaved plasmid DNA vector + DNA fragment from any organism |
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where are restriction enzymes used? |
on the plasmids |
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What are isoschizomers? |
Multiple restriction enzymes can recognize the same sequence
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______________________ bind and cleave ds DNA within or adjacent to a specific sequence called _________________. |
Restriction enzymes; palindromic sequences |
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What are palindromic sequences? |
A DNA sequence that has the same lettering in backwards in the second strand Ex: GAATTC CTTAAG |
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Why are overhangs good?
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Important for DNA complementing |
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What are three patterns of DNA cutting restriction enzymes? |
Sticky ends (5' overhangs) Sticky ends (3' overhangs) Blunt ends |
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The endonuclease hydrolysis is a _________________ reaction and does not require the addition of ATP. |
spontaneous |
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What does Tris-HCl do? |
Prevents degradation and helps with pH |
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List the components of the Restriction enzyme digestion reaction buffer? |
Tris-HCl Divalent cation: MgCl2 Protective carrier protein BSA Salt: NaCl |
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What does BSA stand for as part of the restriction digestion reaction buffer? |
Bovine serum albumin |
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what is the protective carrier protein in the restriction digestion reaction buffer? What does it do? |
BSA; helps the restriction enzyme avoid degredation |
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1 unit = amount of restriction enzyme to digest _____ microgram of reference DNA in ____ hour at 37 degrees celsius. |
1; 1 |
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What does DNA ligase during DNA ligation do? |
Links fragments of DNA together by forming a permanent phosphodiester bond |
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Ligation is a spontaneous reaction that requires addition of ATP and Mg. TRUE OR FALSE |
FALSE; Ligation is NOT a spontaneous reaction that does require the addition of ATP and Mg. |
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Why would you want 2 restriction enzymes rather than one? |
2 REs is better and less risky because with one RE, you're risking the two strands be complementary to one another which could more likely result in a binding not to the fragment. 2 REs would lessen this risk |
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What are the three key elements in plasmids? |
1. Origin of replication 2. Selectable antibiotic resistance gene 3. Multiple cloning site/insertion site/polylinker (lacZ) |
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For the T-easy vector, describe how it works |
In the T easy vector, theres a gap in the lac Z gene that has Ts on both over hangs. Taq DNA will leave As on the overhangs of the fragment DNA making for easy complementary strands when placing fragment in plasmid. |
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Where is the alpha acceptor from? |
The bacteria |
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Where is the alpha donor from? |
From the peptide or vector |
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If the alpha donor and alpha acceptor bind, what is the result? |
beta-galactosidase --> blue colonies of bacteria |
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How does alpha complementation work if the bacteria has the insert?
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The lacZ gene will be interrupted and it kicks out the alpha donor which means you won't get beta-galactosidase = no blue color |
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The blue colonies are bad for alpha complementation. TRUE OR FALSE |
TRUE |
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What is transformation? |
Introduction of plasmid to bacteria
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What is the most common method for transformation? |
Heat shock |
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What are the two methods of transformation? |
Heat shock and electroporation |
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What is the purpose for chemical treatment in preparation of competant E.coli? |
To make bacteria more receptive to plasmid to increase efficiency |
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Describe heat shock |
increase temp to 42 degrees celsius for 1 min. to temporarily open the bacterial wall |
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Describe electroporation |
increase high voltage to open cell wall to insert plasmid |
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Is antibiotic selection enough in the selection of recombinant plasmids? |
No because it only selects for plasmid, not insert |
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In selection of recombinant plasmids, how would PCR help? |
Use primers to detect both plasmids and insert |
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List all the ways you can determine selection of recombinant plasmids (5) |
Alpha-complementation PCR Restriction enzyme digestion Agarose gel electrophoresis Sequencing |
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There are kits that can isolate just the plasmid. TRUE OR FALSE |
TRUE |
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Why do the different forms of plasmid DNA matter?
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Different migration |
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What are the three most common different forms of plasmid DNA? |
Supercoiled DNA Nicked circular DNA Linear DNA |
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Supercoiled DNA runs slow through AGE. TRUE OR FALSE |
FALSE; it runs fast through AGE |
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What could be the reason for there being multiple bands in AGE?
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Different DNA conformations (such as linear, nicked etc) |
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What is a major application for Thermostable DNA polymerase? |
PCR |
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What is a major application for E.coli DNA polymerase I? |
Nick translation |
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To perform a PCR, you have to include the following chemicals in the reaction, including ______a______ (to synthesis new DNA strands), ______b_____ (to initiate DNA synthesis), ____c_____ (to be used as building blocks for synthesis of new DNA strands), ______d________ (to be used as a template for targeted amplification of DNA, and _______e_______ to collectively provide an optimal condition for the enzyme to synthesize new DNA chains. |
a. DNA polymerase b. primers c. DNTPs d. template DNA e. buffer |
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What makes the color change in alpha complementation?
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X-gal |
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Define recombinant plasmid |
insert + plasmid
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Define wild-type plasmid |
Normal circular plasmid WITHOUT insert |
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What is another term for plasmid used in this course? |
vector |
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PCR is primarily a cycling process of three steps: |
1. Denaturation 2. Annealing 3. Extension |
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The insert that is placed in the lacZ gene kicks out the alpha acceptor/deactivates it. TRUE OR FALSE |
FALSE; it kicks out the alpha donor, NOT the alpha acceptor |
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In AGE, sample loading buffer is mixed with DNA samples before loading the sample into the well. Multiple chemicals are present in the buffer for specific purposes. Tris HCl is included to adjust the buffer pH to 8.0 which maintain ______a_______, EDTA is included to minimize ________b__________, glycerol or sucrose is added to help _________c___________, and 2-3 different dyes are included to __________d_____________ and _________e__________. |
a. negative charge
b. DNA degradation c. increase density of sample d. visualize DNA migration e. simplify sample loading process |
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___________ is the alpha donor, and _________ is the alpha acceptor. |
vector/insert; bacteria |
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Is alpha complementation selecting for recombinant bacteria or recombinant plasmid? Why? |
Recombinant plasmid; to tell the difference between wild-type plasmid or recombinant plasmid |
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What selects for recombinant bacteria? |
Anitbiotic resistance gene |
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Define recombinant bacteria |
Bacteria + a plasmid (wild type or recombinant plasmid) |
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When preparing to run AGE it is important to consider how fast the sample will migrate through the gel so that the sample does not run too long. Name four factors that affect the migration rate of DNA through the gel. |
1. Agarose concentration Ex: smaller the pores, slower the migration 2. Size of DNA/amount of sample 3. Voltage strength Ex: weak voltage = slower migration 4. Conformation of DNA Ex: linear vs nicked |
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After obtaining a single PCR product of expected size you send the samples off for sequencing and find that there are several differences between the nucleotide sequence of your gene and the original GAPDH sequence. Is this a problem of specificity or fidelity? List one strategy you could use to reduce the mutation rate within your PCR product. |
Fidelity - increase template and increase cycles |
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Which migrates faster, TAE or TBE? |
TAE |
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What happens if we change pH in PCR? |
Affecting negative charge of DNA |
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Difference between touchdown and hot-start |
Hot start: refers to a type of taq (DNA polymerase); has an antibody that binds to taq at low temps to prevent it from nonspecific amplification
Touchdown (PCR program): refers to how I program my PCR; has to deal with annealing temp; for first few cycles, start with high annealing temp then as cycles progress, the temp will decrease in step-wise fashion |
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Primer dimers can form if our annealing temp is too low. TRUE OR FALSE |
TRUE |
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What three different things can you do to help avoid primer dimers? |
Use touchdown PCR Change primer design Annealing temp |
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Generation of nonspecific DNA products is also very often in PCR reactions. List two different strategies that allow minimizing the appearance of multiple non-specific PCR products. |
Annealing temp Template clean up |
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The time and temp of annealing temp is determined by. |
Melting point |
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When setting up a PCR reaction, these 5 compenents are essential: |
Template DNA, primers, Taq DNA polymerase, dNTPs and buffer |
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This is added to electrophoresis buffer to chelate divalent cations and protect DNA from degradation. |
EDTA |
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Multiple bands in AGE is a problem with what |
decrease in specificity |
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When learning to pipette it is best two aim for what two things? |
Precision and accuracy |
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Difference between Taq polymerase and regular Dna polymerase |
Taq works with 75 to 80 degrees celsius Regular DNA polymerase works with 37 degrees celsius |
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This type of sequences is recognized by RE to cut DNA:
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Palindromic sequences |
