Study sets, textbooks, questions
Upgrade to remove ads
Research Methods Comp Review
Terms in this set (88)
7 basic steps of DNA extraction
4.Remove cellular debris
7.Determine purity & concentration
Why are young plants the best source for DNA?
Younger plants have a higher nuclear volume
to cytoplasmic volume ratio
6 components of lysis buffer
4.Guanidine thiocyanate (salt)
5.DTT (reducing agent)
What is the purpose of guanidine thiocyanate in the lysis buffer?
Denatures proteins and cell membranes via disruption of H-bonding
Aids in DNA silica binding via a cation bridge
You want to load 20 µL into each well for electrophoresis. You use 5X loading dye to add to your initial samples. How much loading dye and sample should you use?
16 µL sample
4 µL loading dye
You have a 40X stock solution. You want a 1X working solution and you need 5 L of it. How much stock solution do you measure? How much dH2O do you dilute it in?
M1(V1) = M2(V2)
40X(V1) = 1X(5L)
V1 = 0.125 L or 125 mL
40X stock solution
5 L - 0.125 L = 4.875 L dH2O
You want to load 24 µL of your nested PCR samples into a gel. You need to load 4X less nested PCR sample than initial PCR sample.
How much 6X loading dye, sterile dH2O, and sample should you use per well?
4 µL 6X loading dye
5 µL nested PCR sample 15 µL sterile dH2O
= 24 µL total
In what direction does DNA Polymerase add new nucleotides?
5' to 3'
Short pieces synthesized from the lagging strand called __________ are joined together by _________.
__________ is used to separate DNA strands in normal DNA replication while _________ is used in PCR.
List the 5 steps involved in DNA replication
1.Template strands separate
2.Supercoiled structure is relaxed
3.Separated strands are prevented from reannealing
4.Short primers of RNA are assembled
5.DNA is synthesized
DNA is ________ charged and will run toward the _______ end.
What can you conclude if you visualize a band in your sterile water negative control lane?
There is contamination.
What is the expected size (base pairs) of the target GAPDH PCR products?
Range from 500 to 2,500 base pairs depending on the plant species chosen
(GAPC 1065 bp & 993 bp)
What is the TAE Buffer used for?
keeps the pH stable and allows for the current to be passed through
How would you be able to distinguish multiple bands of very similar molecular weights?
Increase the percentage of the gel as the slightly smaller band will be able to travel through the smaller matrix more readily
________ primers are used in initial PCR while ________ primers are used in nested PCR.
What are the steps in the PCR cycle?
Why is Taq polymerase important for PCR?
Taq polymerase adds nucleotides to synthesize daughter strands; it is functional at high temperatures (heat stable) and will not be inactivated by denaturation.
Why is exonuclease I used in nested PCR? Explain how it does so.
Exonuclease I removes primers from the first round that were not incorporated.
It digests only single-stranded DNA.
What are 6 factors to consider in designing primers?
What is the desired gene we are targeting throughout this entire experiment?
What are the 3 buffers used in PCR purification?
Buffer PB, PE, & EB
Why do we blank the NanoDrop and what do we blank it with?
To establish background wavelength
Whatever the sample was eluted with (water or EB buffer)
What equation can be used to determine the # of pieces of exact size dsDNA in PCR?
2n - 2n
(where n = PCR round #)
What well-known model organism/species do we utilize in our experiments and why is it used as a model system?
It has a rapid life cycle (6 weeks), small genome (125 Mb), and prolific seed production.
What is the nutrient solid media used for propagation & maintenance of E. coli?
Why is streaking used?
To dilute the # of cells and increase the likelihood of producing a single colony (genetically identical)
What sterile techniques were used in media prep?
This selects for bacteria containing plasmids with the resistance gene.
IPTG activates ________ to facilitate gene expression on the plasmid.
What is this ligation product?
Ligation of 1 insert into vector
What vector did we use and what is its approximate size?
In DNA ligation with _____ ends, there are overhangs on the end of DNA strands.
What fuses ends of the vector to ends of the PCR product/insert?
T4 DNA ligase
Ideally, what 2 ligation products would we not see on our plates and/or gels?
Self-ligation of vector
Self-ligation of insert
What do we want on our plates after transformation?
A single colony
What 2 methods of transformation were discussed?
Which was used in this experiment?
What would we expect to see on the negative control plate? (competent cells only on LB/AMP/IPTG)
List the 6 basic steps involved in making competent cells.
1.Incubate starter culture in C. growth media
3.Keep pelleted cells on ice
4.Add transformation buffer
5.Incubate on ice
6.Repeat steps 2-5
What do the calcium chloride and DMSO do in the transformation buffer when making competent cells?
Calcium chloride: causes DNA to bind to bacterial cell wall
DMSO: assists the plasmid DNA to pass through lipid cell membrane
How do we provide oxygen to bacteria?
What restriction enzyme was used and what type of digestion does it produce?
Sticky end digestion
Name the 5 solutions used in plasmid purification.
What is the desired result on a gel after digestion?
One around 1000 bps
One around 3000 bps
(vector and insert separate)
Why might there be more than 2 bands in one lane on a gel after digestion?
The insert contains a Bgl II site
What type of sequencing did the Keck facility perform?
BigDye Terminator Sequencing
What do the sequence of peaks in a chromatogram represent?
The sequence of bases in a DNA molecule
Name 3 differences between PCR and the sequencing we used.
•PCR uses 2 primers to amplify gene; sequencing uses 1 primer to obtain DNA sequence
•Sequencing uses dye-labeled ddNTPs
•Run through capillary system to generate sequence
List 7 things that go inside the sequencing reaction.
What are ddNTPs and how do they work?
Dye-labeled nucleotides that do not have 3'OH group (modified so there is an H) so when incorporated into DNA, synthesis will end at that nucleotide
How many/what plates did you pour per sample?
4 plates total:
1 LB only
What type of DNA ligation did we use?
Blunt end ligation
What is toxic to E. coli when expressed?
Describe the shape of supercoiled circular plasmid DNA and how it might run on a gel.
moves through gel faster (can appear smaller than plasmids cut by restriction enzymes)
What is the role of proofreading polymerase and for what experiment did we use it?
Removes the 3'-A left by Taq, leaving blunt ends
Used in ligation
What was constructed in order to determine the protein concentration from the absorbance data?
What is in each layer after centrifugation in the protein extraction?
top- organic layer
What was used in the protein extraction to prevent product degradation?
Halt Protease Inhibitor Cocktail
List 3 advantages of using a BCA assay.
•Color complex is stable
•Less susceptibility to detergents
•Can detect a broad range of protein concentrations
Using the following equation, calculate how much sample to load if we wanted 50 ug.
y = 0.0003x + 0.017
Absorbance = 0.709
0.709 = 0.0003x + 0.017
x = 2306.67 ug/mL
2036.67x = 50
x = 0.02168 mL
= 21.68 uL
What is the purpose of bromophenol blue?
What type of gel is used in SDS-PAGE & why?
Can withstand the high voltage necessary to create the charge gradients
Has pore sizes similar to size of proteins (gel matrix much tighter than agarose)
What is the role of
Reducing agent: disrupts disulfide bonds (3o & 4o structures)
Describe the mechanism of tri-halo compounds.
UV light → compounds irreversibly crosslinks with tryptophan residues on samples → produces fluorescence
What does SDS do & how does it accomplish this?
•Makes proteins negatively charged (2- charges per SDS molecule)
•Denatures 2o & non-disulfide linked 3o structures
•Has a hydrophobic tail that interacts strongly with protein chains
What type of transfer did we use?
___________ is the term for detecting multiple proteins on a blot using 2 distinct dye-labeled 2o antibodies.
Describe the difference between direct & indirect methods of Western Blotting.
Direct - enzyme- or fluorophore-conjugated 1o antibody
Indirect - unlabeled 1o antibody + enzyme- or fluorophore-conjugated 2o antibody
Describe 2 differences between PVDF & nitrocellulose membranes.
Which one did we use?
•PVDF is highly hydrophobic, must be pre-wetted with methanol or ethanol
•PVDF is less brittle (can be used for stripping and re-probing)
Nitrocellulose was used
Describe the process of creating 1o antibodies & explain how to determine an appropriate 2o antibody.
1o - protein injected into animal; antibodies produced & extracted → recognize target protein
2o - need to consider host species of 1o antibody (anti-host species)
What type of experiment are knock-out & blocking experiments?
Correlation does not imply causation. What experiments are important to scientifically establish causation?
Necessity & Sufficiency
What type of experiment are rescue & overexpression experiments?
What is the difference between the independent & dependent variables?
Independent - condition of the experiment that is manipulated by the experimenter
Dependent - what is being observed/measured under the different experimental conditions
During SDS-PAGE, we were left with linear, negatively charged polypeptide chains. What caused this?
What loading control was used in Western Blotting? Why?
Indicates that the samples were loaded equally
If your goal was to isolate and clone a gene from a plant in order to determine the specific gene sequence, what experimental tasks (in order) would be completed?
1.Extract DNA from plant
2.Amplify region of gene by PCR
3.Analyze, purify, & quantify the PCR product
4.Ligate PCR product into cloning vector
5.Transform bacteria with plasmid
6.Isolate & sequence plasmid
7.Analyze cloned gene with bioinformatics
What is the role of the wash buffer in Western Blotting & what is it composed of?
•Removes unbound reagents; reduces background
•Tris buffer saline + tween 20 (TBS-T)
Reduction of ____ to ____ is proportional to protein present in the BCA Assay.
Cu2+ to Cu1+
How much antibody (2 mg/mL stock) do I use in 15 mL of blocking buffer if I want a working concentration of 1 ug/mL?
M1(V1) = M2(V2)
2 mg/mL (x) = 1 ug/mL (15 mL)
2 mg/mL (x) = 0.001 mg/mL (15 mL)
2x = 0.015
x = 0.0075 mL
x = 7.5 uL
What was the purpose of BSA in the blocking buffer?
To prevent the antibodies from binding to the membrane
Is having the flu virus in your blood a necessary or sufficient condition for being sick?
Having the flu virus is sufficient for being sick, but not necessary since there are other ways to be sick
What is the difference between the stacking & resolving gel?
Stacking gel - concentrates proteins together in gel so they enter the resolving gel at the same time
Resolving gel - separates proteins by size
What is the arrangement of the blotting sandwich for the Semi-dry transfer?
Top (-) cassette electrode (cathodes)
Top ion reservoir stack
Bottom ion reservoir stack
Bottom (+) cassette electrode (anode)
What is the full name of GAPDH?
To prepare a working reagent, I need to mix 50 parts Reagent A with 1 part Reagent B (50:1, A:B).
If I require 8 mL, how much of each do I add?
8,000 uL / 50 = 160 uL
8 mL Reagent A
160 uL Reagent B
(Do not subtract the 160 uL from the 8 mL! This is a ratio, not a dilution!)
Sets found in the same folder
Histology Exam 1
Histo Exam 2
Histo Exam 3
Other sets by this creator
MB Exam Jen Questions
MC ASCP- Set D
MB ASCP- Set C
Other Quizlet sets
Health Care in Special Populations II
Fluid and Electrolyte, and Acid-Base Balance