M3 (PART 3 AND 4): NUCLEIC ACID EXTRACTION Flashcards
at what temperature is proteinase K (biospin bacteria genomic DNA) stored at ?
2-8C
What is the temperature of the other components (except proteinase K) in the biospin kit stored at ?
15-25C
In general, the biospin kit can get at most __ genomic DNA from ___ bacterial cells
30ug
5x10^9
How long does it take for the biospin kit to isolate high yields of pure genomic DNA ?
less than one hour
What are the advantages of the biospin kit for bacteria Genomic DNA extraction ?
simple fast very economic no expensive equipment few steps avoids use of toxic and hazardous reagents (phenol chloroform)
In biospin bactreria genomic DNA kit, what is the purpose of the EL buffer ?
completely destroys the structure of bacterial cells
These components of the biospin bacteria genomic DNA kit allows the DNA in the sample to be liberated
RS Buffer
GA Buffer
PK solution
In biospin bactreria genomic DNA kit, released DNA is bound exclusively and specifically to the ____ in presence of a ____ under appropriate ___ and ____ conditions
biospin membrane
binding buffer
salt iron
pH
What are additional steps to take note of when handling with gram positive bacteria ?
add 158 mg Lysozome to EL buffer
Mix thoroughly for 30 seconds or so until solution is clear
store at 2-8C
It is important to add ___ to BA buffer and mix thoroughly before use
10.5ml absolute ethanol
It is important to add ___ to Wash Buffer and mix thoroughly before use
31.5 ml absolute ethanol
In order to optimize the effective result, the appropriate number of bacterial cells is at most ____, which OD 600 is between ___
5 x 10^9
1.0~2.0
What to do if RS buffer forms precipitates upon storage ?
incubate the buffer at 56C until precipitate has been fully dissolved
What is the incubation protocol for gram negative bacteria ?
37C for 10-15 minutes
What is the incubation protocol for gram positive bacteria ?
37C for 30-60 minutes or more times
What is the protocol for lysis of the sample ? What to do if is difficult to be lysed ? in biospin
56C for 15 minutes
extend incubation time
What is the protocol to be followed if there are mucous material present in the supernatant ?
transfer them to a new 1.5ml tube
What can we do if RS buffer solution turn to muddiness in biospin bacteria genomic dna test kit ?
shake up thoroughly
Is RNAse A necessary for the test for biospin bacteria genomic dna test kit ? ?
determined by test aim
not necessary, if the extracted genomic DNA is used to backward test of molecular biology such as PCR, enzyme digestion, and so on
necessary for RNA digesting adequately, if we want to get high purity genomic DNA
Is there organic solution in the extraction process of biospin bacteria genomic dna test kit ?
No
How long about the extracted bacteria genomic DNA fragments from biospin bacteria genomic dna test kit ?
Among 20-150KB
What’s the matter about the small quantity of the extracted genomic DNA of biospin bacteria genomic dna test kit ?
Bacteria is in the position of logarithmic phase (when bacteria reproduce fast)
Add lysozome in EL buffer and shake up sufficiently
After adding EL buffer solution, break up the bacteria mass at the bottom of the tube and provide enough incubate time at 37C
Ensure that other extraction processes follow with the specification strictly
What’s the quantity range of the bacteria for extraction of biospin bacteria genomic dna test kit ?
quantity is among 10^7~10^8/ml
incubated on liquid culture medium
logarithmic phase
maximum quantity: 5x10^9
What are the four major factors to consider when selecting extraction method ?
type of nucleic acid
source of nucleic acid
type of downstream application
sample batch size and processing time
What are the fundamental steps of nucleic acid extraction ?
cell lysis
separation of DNA from cellular debris and other cellular components
alcohol precipitation
During cell lysis ____ is essential as these proteins can degrade nucleic acid and interfere with the analysis
denaturation of proteins
What does the cell suspension tube contain ?
intact cells
What does the cell lysate tube contain ?
nucleic acids and other cell components
This cell lysis method utilizes detergents and chaotropic agents to disrupt cellular membrane and denature proteins
Chemical method
This cell lysis method cellular membrane and denature proteins
Enzymatic method
This cell lysis method includes freeze-thaw cycles, mechanical lysis, liquid homogenization, sonication, or high pressure to lyse cells that are hard to disrupt.
Physical method
How does vigorous sample manipulation such as prolonged vortexing affect the yield of cell lysis ?
may shear nucleic acid
Why must cell lysis be performed as quickly as possible ?
to avoid nucleic acid degradation
This liquid phase extraction method can separate nucleic acid from other cellular components based on their differential solubility
Organic extraction
What organic solvents are used in liquid phase organic extraction method ?
phenol and chloroform
A biphasic emulsion can be found in which liquid phase extraction method ?
Organic extraction
What does the lower phase of the liquid phase organic extraction method contain ?
organic solvent phase
lipids, denatured proteins, and other cellular components
What does the upper phase of the liquid phase organic extraction method contain ?
aqueous phase
nucleic acid
In organic extraction what pH is DNA extraction performed in ?
pH 7-8
In organic extraction what pH is RNA extraction performed in ? and in which phase is the RNA found in ?
pH 4-6
RNA = aqueous phase DNA = organic phase
What is added to achieve the acidic pH for organic RNA extraction ?
2 M sodium acetate solution
____ is commonly included in organic RNA extraction to help reduce RNase activity
guanidinium thiocyanate
What are the benefits of organic extraction of DNA ?
Rapid denaturation of proteins
Efficient extraction of high molecular weight nucleic acid
Suitable for various cells and tissue types
What are the drawbacks of organic extraction of DNA ?
Exposure to hazardous organic reagents
Labor-intensive procedures
Difficult for automation
This liquid phase extraction method is also known as “salting out”
Inorganic extraction
Inorganic extraction leaves DNA in the solution by using high concentrations of salt such as ___ to selectively precipitate proteins
ammonium acetate
potassium acetate
sodium chloride
What are the benefits of inorganic extraction of DNA ?
No hazardous organic solvents
Simple protocol
Economical method for routine use
What are the drawbacks of inorganic extraction of DNA ?
Time consuming process when using a traditional salting out protocol
Incomplete removal of salt causing interference with nucleic acid analysis
This solid phase extraction method employs selective binding of nucleic acid to a solid matrix such as silica that is packed in a column
Column-based extraction
In column-based extraction the passing of a buffer containing ___ through the column to remove nucleic acid-binding proteins may stay bound to nucleic acid
low amount of chaotropic salt
In column-based extraction of DNA applying this to wash out contaminants improves the purity of the alluded nucleic acid
wash buffer
In column-based extraction of DNA, applying this will release the nucleic acid from the solid matrix into a collecting container
elution buffer or water
What are the benefits of column-based extraction of DNA ?
Convenience and ease of use
Scalability
Ability to automate
What are the drawbacks of column-based extraction of DNA ?
Propensity for clogged matrix
Fixed nucleic acid binding capacity
Binding is dependent upon nucleic acid size
This solid phase extraction method utilizes small particles with a paramagnetic core that binds to nucleic acid
Magnetic bead-based extraction
This extraction method is commonly used in automated nucleic extraction systems
Magnetic bead-based extraction
What are the benefits of magnetic bead-based extraction of DNA ?
No risk of column clogging
High nucleic acid binding efficiency
Rapid collection or concentration of sample
Ability to automate
What are the drawbacks of magnetic bead-based extraction of DNA ?
Potential carry-through of magnetic particles into final samples
Slow migration of magnetic particles in viscous solutions
Often relies on automated machinery
In the removal of contaminants this is used when the sample volume is large
Isopropanol
Which is used to remove salts in the removal of contaminants and nucleic acid recovery ?
ethanol
Which is used to precipitate nucleic acid in the removal of contaminants and nucleic acid recovery ?
isopropanol
In the analysis of nucleic acid quantity and purity this is a method that measures the amount of light absorbed by nucleic acid in samples.
Spectrophotometric measurement
In the analysis of nucleic acid quantity and purity this is a method that resolves nucleic acid on a gel matrix based on their size and charge.
Gel electrophoresis
This is the most commonly used method for determining nucleic acid concentration
Spectrophotometric measurement
What is the constant for dsDNA when calculating the estimate of nucleic acid ?
50
What is the constant for ssDNA when calculating the estimate of nucleic acid ?
33
What is the constant for RNA when calculating the estimate of nucleic acid ?
40
Protein contamination in a nucleic acid sample can be indicated by the ratio of UV absorbance at ___ and ___
260 nm
280 nm
In general, samples with good purity are DNA samples with an A260/A280 ratio of
1.7-1.9
In general, samples with good purity are RNA samples with an A260/A280 ratio of
≥2.0
What are the advantages of using spectrophotometry measurement ?
Simple and fast method
Easy sample preparations
What are the disadvantages of using spectrophotometry measurement ?
No information about DNA and RNA cross-contamination or nucleic acid integrity
Interference of A260 from contaminants
Relatively insensitive detection as it requires 5 μg/ml of dsDNA to observe an A260 of 0.1
A method used for the separation of nucleic acid used on a porous gel matrix
Gel electrophoresis
These type of gels are typically run vertically, where nucleic acid separation is based on size, shape, and charge
Polyacrylamide
These type of gels typically run horizontally, where nucleic acid separation is based only on size
Agarose
What net charge does a nucleic acid carry ?
negative
Between small and larger nucleic acid fragments, which one is observed near the positive end and the negative end in gel electrophoresis ?
positive end: smaller nucleic acid fragments
negative end: larger nucleic acid fragments
Why is it important important to ensure that the electrodes are oriented in the correct direction before you run gel electrophoresis ?
The electric field will be reversed if the electrodes are reversed leading to nucleic acid migrating into a wrong direction
What will happen to the results if gel electrophoresis running time happens for a long time ?
can cause smaller nucleic acid fragments to run off the gel
What is the commonly used stain in gel electrophoresis ?
ethidium bromide
What is the lower toxicity and higher sensitivity alternative stain used in gel electrophoresis ?
SYBR Green
This is used for the quantification of nucleic acid in gel electrophoresis
gel densitometry
Protein bands are not visible by ethidium bromide staining in gel electrophoresis, what can indicate protein contamination ?
retention of nucleic acid in the well
a smeared band
What may be the reasons to a smeared band in gel electrophoresis ?
protein contamination
nuclease activity
too much salt in the sample
What are the advantages of using gel electrophoresis ?
Physical separation of nucleic acid from some contaminants.
Potential identification of unwanted nucleic acid.
Purification of nucleic acid from the gel for downstream applications.
What are the disadvantages of using gel electrophoresis ?
A complicated technique required for separation of DNA larger than 20 kb.
Possible exposure to mutagenic agent if ethidium bromide is used as the stain.
Exposure to UV light upon band visualization
What is the different between prepared and pre-cast gels in gel electrophoresis ?
Prepared gels are less expensive, can be tailored to your exact specifications, and can be prepared as needed.
Pre-cast gels provide convenience and time savings, reduction of exposure to toxic gel reagents, gel quality consistency, and reproducibility in data
In organic extraction of DNA exposure to this is through the skin can cause severe burns and systemic effects.
phenol
In organic extraction of DNA this is skin and eye irritant and a suspected carcinogen.
Chloroform
What are the factors to consider to prevent DNA degradation ?
Acidic conditions can render DNA hydrolysis. A 10 mM Tris buffer (pH 8-9) is typically used for DNA storage where the downstream applications allow.
Ethylenediaminetetraacetic acid (EDTA) is also commonly included in the storage solution to inhibit DNase activity where the downstream applications allow.
Large DNA molecules such as genomic DNA can break easily. Physical manipulation such as excessive pipetting and vortexing should be avoided.
Improper storage temperature can lead to DNA degradation. DNA can be stored at 4°C for short-term storage. For long-term storage, DNA should be divided into small aliquots and stored at -20°C or -80°C to avoid repeated freeze-thaw cycles.
What are the factors to consider to prevent RNA degradation ?
Wearing gloves when handling RNA at all times
Using RNase-free reagents and disposables such as tubes and barrier tips.
Adding additives such as RNase inhibitors in the lysis buffer when possible.
Decontaminating glassware and the work area.
What is the troubleshooting for low nucleic acid yields ?
Low quality of tissue or cell sample. Proper treatment and storage of biological samples prior to extraction is essential for preserving nucleic acid.
Incomplete lysis. Complete lysis is essential for successful release of nucleic acid from the cells. Insufficient lysis could be due to an incorrect protocol or insufficient amount of lysis buffer.
Incorrect extraction solutions. When the buffers are not prepared correctly, nucleic acid may migrate to a different liquid phase or may not bind to the solid phase.
Incorrect elution solution. In solid-phase extraction, the compositions and volume of elution buffer can greatly affect product yields.
In troubleshooting for purity of nucleic acid what does a strong A280 indicate ?
contaminants such as proteins.
In troubleshooting for purity of nucleic acid what does a strong A230 indicate ?
contamination of organic compounds, ethanol, or salts.
In troubleshooting for purity of nucleic acid what does a strong A270 and A275 indicate ?
phenol contamination
How to reduce instances of a strong A230 ?
An additional step of alcohol precipitation or ethanol wash may be required
Residual ethanol can be removed by increasing the drying time of the nucleic acid pellet
How to reduce instances of a strong A270 and A275 ?
repeating alcohol precipitation or using a column-based nucleic acid clean up kit.
Why is degraded DNA not suitable for downstream processes like PCR ?
as the process might fail because the target gene might be cut off
If there are no bands in the gel electrophoresis, the process must be repeated with some precautions, which are ?
Correct handling and storage of starting materials
Perform extraction at 4C or on ice
Inhibit nuclease activity
Store purified DNA correctly
Use sterile tips and tubes
Pipetting errors
