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What is the purpose of the very first centrifugation during the plasmid mini-prep protocol
To pellet intact bacterial cells in the culture of the tube.
What enzyme was pre-mixed with Solution I (the solution that the pelleted bacterial cells are resuspended in)?
After adding Solution II (the cell lysis solution), one wants to shake the tube vigorously.
Solution III precipitates protein. Therefore, after adding solution III and centrifuging, we want to transfer the ___ and apply it to the HiBind DNA Mini Column.
Prior to usage in class, the HBC buffer was diluted with:
Prior to usage in class, the DNA Wash Buffer was diluted with:
After doing a total of two washes with the DNA wash buffer, the next step was to:
Dry the DNA binding column by spinning it empty for two minutes.
The DNA bind column is placed in to a new 1.5 mL microfuge tube before adding the elution buffer.
If we have a really large plasmid (greater than 10 kb), we can better DNA elution off the DNA binding column if we preheat the elution buffer to 70C before applying it to the DNA binding column.
After centrifugation of the eluted DNA, it should be stored at __ degrees Celsius.
Whether grinding up the frozen plant tissue with either a mortar and pestle or a grinding mill, the ground tissue should be transferred while still frozen to Lysis Buffer A.
There is a second lysis buffer, Lysis Buffer B, that's added after Lysis buffer A. It has the enzyme ___ already added to it.
After both lysis buffers were added, at what temperature were the samples incubated for 10 minutes with occassional mixing?
What is the precipitation solution precipitating?
Proteins and polysaccharides
Precipitation is assisted by incubating the samples:
An equal volume of plant gDNA binding solution and __ is added to the supernatant resulting after ridding the sample from protein and polysaccharides, before loading the sample on the DNA purification column.
After washing the sample with each of two wash buffers which have already had ethanol added to them, the genomic DNA will be eluted in water.
If one is extracting genomic DNA from plant material that is rich in lignin, Lysis Buffer A is used supplemented with __ at a 2% (w/v) final concentration.
All pipetting and vortexing steps should be done as gently as possible.
One should only use up to __ mg of fresh or frozen tissue (By the way, both types still contain water; which is why using more than the 20 mg max for lypholized- freeze dried tissue) is ok.
By adding 5 ul of dNTP Mix (2 mM each) into the PCR reaction that will have a total volume of 50 ul, what will the final concentration of each dNTP be?
DreamTaq Hot Start DNA polymerase, like most Taq (Thermus aquaticus) DNA polymerases, generates 3' -dA overhangs on both ends of the PCR product.
If more than 10 copies of template DNA are present in the PCR reaction, how many cycles of PCR are sufficient?
If one is trying to amplify a DNA template that has a GC content greater than 60%, how should one adjust the initial DNA denaturation from the temperature or time normally used?
Keep the denaturation temperature at 95C, but lengthen the denaturation time longer than 3 minutes
If one runs their PCR reactions on an agarose gel, and sees that they got non-specific PCR products made due to mis-annealling of the primers, how do you think they should adjust their cycling temperature conditions when they repeat their PCR reaction?
Raise the primer annealling temperature in 1-2 degree increments
The final concentration of primers in the PCR reaction is 0.1 to 1 uM. What is the risk of excessive primer concentrations?
Some primer molecules anneal to the wrong places on the template DNA, generating non-specific PCR products.
The recommended template amount range in these PCR reactions is 10 pg to 1 ug, in general. In the case of using genomic DNA as template, the protocol recommend 100 pg to 1 ug added into the PCR reaction. What is the risk in adding too much template DNA?
Increases the risk of getting non-specific amplification (the primers don't just anneal and allow amplification of the correct product, but also mis-anneal to wrong places on the template DNA and amplify non-targeted regions)
Using DreamTaq DNA polymerase, the primer annealling temperature should be:
Equal to the primers Tm
We are planning to purify our PCR amplified gene promoters and then TOPO TA clone them into the PCR8 GW TOPO Gateway Cloning entry vector, which comes linearized with T overhangs. Why should we make sure we do a 15 minute 72C incubation after the last PCR cycle?
Allow time to fill in any possible incomplete reaction products
Why can we set up our PCR reactions at room temperature instead of on ice when using a Hot Start DNA polymerase, yet still get specific amplification?
Hot start DNA polymerases include an antibody in with the DNA polymerase that prevent its polyermase activity at ambient temperatures, but the antibody releases at polymerization temperatures.
Because Cas9 will keep causing double stranded breaks as long as the gRNA target site/PAM site remain intact, it is wise to block further editing of the desired HDR repair events by:
Incorporating into the HR template a mutated PAM or gRNA seed sequence
Before the ends can be ligated together during NHEJ, what needs to happen at the double stranded break ends?
Missing or damaged nucleotides need to be processed by an array of processing factors(including polymerases, nucleases, and structure-specific end cleaning enzymes) to make ends that are better substrates for ligation
Comparing Non-Homologous End Joining and Homology Directed Repair double stranded break mechanisms, which mechanism finishes repairs of most types of breaks first?
During NHEJ, which two proteins form a heterodimer that functions in DNA repair by first recognizing the broken DNA ends, and then serves as a scaffold for recruitment of a kinase and a 2 subunit DNA ligase to form a complex that holds the DNA ends together, forming a paired end complex?
Ku70 and Ku80
If one wants to insert sequences longer than 200 bases by homologous repair, one should use __ as the source of the homologous DNA repair template:
Double stranded DNA plasmid templates
In the __ mechanism to repair HDR induced strand invasion and D-loop formation, the newly synthesized sequences are present on only one of the two resulting DNA molecules.
Synthesis-dependent stand-annealing (SDSA)
In order for Homology Directed Repair mechanism to occur, a repair template needs to be present, and this repair template needs to have regions of DNA with sequence homology to:
DNA sequence on either side of the Cas9-induced stranded break
In the classical double stranded break-repair pathway of HDR, what are the FOUR-STRANDED BRANCHED STRUCTURES called that form when elongation of the invasive strand "captures" and synthesizes DNA from the second double stranded break end?
double Holliday junctions (dHJs)
In the first step of the classical double stranded break-repair pathway of HDR, 3' ended overhangs have to be generated so that they can invade the intact homologous DNA template and serve as a template for DNA repair synthesis.
NHEJ repair normally occurs much more frequently to repair double stranded breaks over homology directed repair (HDR), even when a donor template has been provided. Which of the following strategies have been used successfully to promote HDR over NHEJ:
Introducing protein factors that activate HDR
Introduce NHEJ inhibitors
Use Cas9 fused to a protein that both inhibits NHEJ and activates HDR
*All of these
One can use 2 CRISPR guide RNAs to target 2 Cas9 proteins hundreds of base pairs apart to generate 2 double stranded DNA breaks, with the goal of the long DNA sequence located between those 2 DSBs being deleted during the subsequent NHEJ repair.
Since NHEJ only incorrectly repairs a DNA double stranded break incorrectly less than 5% of the time, why is it that scientists using CRISPR/Cas9 are able to obtain many instances of INDELS (insertions and deletion mutants) created by NHEJ incorrect repair events?
Because Cas9 will repeatedly generate DNA double stranded breaks at the correctly repaired targeted DNA sequence, until it has in fact been incorrectly repaired.
The strategy of using 2 guide RNAs in combination with two mutant Cas9 nickases (where each nickase cuts only 1 DNA strand located opposite each other), offers an advantage over using one guide RNA to target normally functioning Cas9 to create a double stranded break at a targeted location. What is the advantage of using 2 guide RNAs targeting 2 Cas9 nickases on opposite DNA strands very close together?
Greatly reduces off-target effects: getting double stranded breaks and repairs at non-targeted sites in the genome
What is the approximate chance of a frameshift inducing mutation being induced during NHEJ DNA repair?
Two-thirds (66.67 % chance)
What is the Spike Control used for in an ELISA assay?
To compare to the standard to detect other proteins that might affect your ELISA assay
What lets you know that your protein of interest is detected in your ELISA assay?
The antibody causes the substrate attached to the epitope to change colors.
What calculations should be used to interpret your data correctly?
Curve-fitting software to generate a standard curve
Take into account dilution factors
Calculate average, SD, and CV on replicates
ALL OF THE ABOVE
I mentioned 4 different types of ELISA assays all used for different interpretations of antibody detections:
ELISA assay can be used to detect COVID-19 virus antibodies (even the new strain that has mutated), and help scientists have more control over the pandemic.
What are some of the advantages of BiFC?
Absence of background signal
BiFC is a relatively hard method to do and it needs specialized equipment to perform it
False, it is relatively simple, and only needs wide field fluorescence or confocal microscope to check for fluorescence signal.
What controls do we use to make sure that the fluorescence being seen is due to correct protein interaction?
Mutant protein should be fused to the fluorescent protein fragment in the same way to the wild-type protein
YFP are highly recommended to use in BiFC?
Based on the review article, which of the following is not necessary for BiFC?
DNA Micro array is used to screen patients for patterns of genes in a particular condition or disease
What are the applications that DNA microarray can be useful?
Correlating gene expression with metabolic changes
Characterizing cell types
ALL OF THE ABOVE
The first step in the process of DNA microarray is to reverse transcribe and label the mRNA
What is the control group in the DNA microarray experiment?
The known sample of blood containing said gene
The tags on the bound cDNA are excited by using a laser and the labeled target that bind to the probe generate a signal
When performing the crosslinking step of ChIP-Seq, what is the most common fixative used?
The purpose of adding glycine to formaldehyde in the crosslinking step of ChIP-Seq is to quench the formaldehyde
The positive control in ChIP-Seq is used to determine background (signal: noise)?
Which method is more suitable for transcription factors/cofactors in the chromatin fragmentation step of ChIP-Seq?
In the immunoprecipitation step of ChIP-seq, which protein beads are the most ideal for most labs?
RFLP is used for identifying point mutations, genome mapping cut is costly so it is losing popularity.
Researchers used PCR techniques with RFLP to accomplish:
Amplifying KDR fragments of VSSC gene
RFLP was discovered by Alec Jeffery while researching pattern of inheritance.
While using RFLP researchers learned their samples had _______ point mutations, which could be seen using _______ which reveals specific banding patterns.
2; Gel electrophoresis
When researching resistance in head lice, the process of identifying point mutations began with:
Using PCR technique to amplify the XDR fragments
What sequence does the EcoRI cleave?
The smallest working genetic sequence allows a gene to express itself is the base elements.
Proteins normally bind to the ______ and will either express or repress _______
It is not important to run a PCR when using a promoter bashing assay.
What sequence does the HindIII cleave?
What two scientists created the Yeast-Two Hybrid Assay?
Stanley Fields and Ok-kyu Song
The Ptashne Laboratory published their discovery of the modular structure of a transcriptional activator in yeast: Gal4 which inspired the creation of the Yeast-Two Hybrid Assay
What was the bait protein used in the Ebola Yeast-Two Hybrid Assay?
What does DBD stand for as it pertains to a Yeast-Two Hybrid Assay?
DNA binding Domain
The yeast saccharomyces cerevisiae was studied during the creation of the Yeast-Two Hybrid Assay
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