Lab 2: Extraction Of RNA, DNA and Protein Flashcards
What method is used for RNA isolation from peripheral blood leucocytes?
Acid phenol guanidine thiocyanate method
This method lyses white blood cells using guanidine thiocyanate in the presence of acid phenol.
What happens to DNA and RNA during the phase separation in RNA extraction?
DNA partitions into the organic phase; RNA remains in the aqueous phase
This occurs after the lysis of leucocytes and phase separation in the extraction process.
What is the first step in the extraction of total RNA from peripheral blood leucocytes?
Add 300ul of whole blood to 900ul of red blood cell lysis solution
The lysis solution contains 155mM Ammonium chloride, 10mM Potassium Hydrogen Carbonate, and 1mM EDTA.
What is the incubation time for the blood and lysis solution mixture?
10 minutes
The mixture is incubated at room temperature and inverted occasionally.
What is the purpose of centrifuging the sample after incubation with the lysis solution?
To form a white pellet of unlysed leucocytes
The supernatant contains lysed erythrocytes.
What reagent is added to the resuspended leucocyte pellet after centrifugation?
500ul of Tri reagent
Tri reagent facilitates RNA extraction by lysing the cells.
What is added to the cell suspension after the Tri reagent, and what is its purpose?
50ul of bromochloropropane; to aid in phase separation
This step is crucial for effective extraction of RNA.
At what temperature should the centrifugation occur after adding bromochloropropane?
4°C
This temperature helps to stabilize the RNA during the extraction process.
What is done with the upper (aqueous) phase after centrifugation?
Transfer to a fresh tube and add an equal volume of isopropanol
This step precipitates the RNA from the aqueous phase.
What is the purpose of adding 1ml of 75% ethanol after RNA pellet formation?
To wash the RNA pellet
This step helps to remove impurities and residual salts.
What is the final step in resuspending the RNA?
Resuspend in 25ul of RNase free water and heat to 65°C
Heating helps to dissolve the RNA effectively.
How is RNA concentration estimated after resuspension?
Using a UV spectrophotometer
This method provides a quantifiable measure of RNA concentration.
What is the purpose of adjusting the pH of the solution in DNA extraction?
To allow DNA to partition to the aqueous phase
This is typically done with 1M Tris base.
What components are included in the back extraction buffer for DNA extraction?
- 4M guanidine thiocyanate
- 50mM sodium citrate
- 1M Tris
These components help in the extraction process of DNA.
What is the first step after adding back extraction buffer to the retained organic phase?
Vortex and retain for 10 minutes at room temperature
This step ensures thorough mixing of the buffer with the organic phase.
What is the centrifugation time and temperature for the DNA extraction process?
5 minutes at full speed in a benchtop microfuge at 4°C
This step separates the aqueous phase from the organic phase.
What is added to the aqueous phase after centrifugation in DNA extraction?
300ul isopropanol
This aids in precipitating the DNA.
What should be done with the organic phase after transferring the aqueous phase?
Retain it for Protein extraction
The retained organic phase contains proteins that can be extracted.
How is the DNA pellet washed after centrifugation?
With 70% ethanol
This wash step helps to purify the DNA pellet.
What is the final step in resuspending the DNA pellet?
Resuspend DNA in 50ul of TE and incubate at 65°C for 1 hour and overnight at room temperature
This helps to dissolve the DNA for later analysis.
What is the method used to estimate DNA concentration?
UV spectrophotometry
This technique allows for quantification of DNA concentration.
What is added to the retained organic phase for protein extraction?
750ul of isopropanol
This is the first step in the protein extraction process.
What is the washing solution used for protein pellet washing?
0.3M guanidine hydrochloride in 95% ethanol
This solution is used to clean the protein pellet.
What temperature and duration are required for boiling the protein sample loading buffer?
100°C for 5 minutes
This step prepares the protein for analysis.
At what wavelength does DNA absorb UV light?
260nm
This absorption is used to determine DNA concentration.
At what wavelength do proteins absorb UV light?
280nm
This is used to assess the purity of DNA solutions.
What is the significance of the ratio between the absorption at 260nm and 280nm?
It indicates both the concentration and purity of DNA
A pure DNA solution should have a 260/280 ratio greater than 1.7.
What should be done before measuring DNA concentration with a nanodrop?
Blank the nanodrop using water
This ensures accurate measurements.
What is the procedure for measuring DNA concentration using a nanodrop?
1) Blank with water
2) Replace water with 2u of DNA solution
3) Read absorption at 260nm and 280nm
This is a standard practice for spectrophotometric analysis.
What does an A260 value of 1 indicate in terms of DNA concentration?
50ug/ml
This is a standard conversion used in DNA quantification.
How is the test DNA concentration determined using the A260 reading?
A260 X 50 X 100 = concentration in ug/ml
This formula helps convert absorbance to concentration.
What is the typical storage temperature for DNA samples?
-80°C
This is recommended to preserve DNA integrity.
What is the expected A260 and A280 reading for a 50.00 ng/ul DNA solution?
A260: 16.411, A280: 8.861
These values are indicative of the DNA concentration and purity.
Fill in the blank: A pure DNA solution should have a 260/280 ratio greater than ______.
1.7
This ratio is crucial for determining DNA purity.
True or False: Proteins can interfere with the measurement of DNA concentration at 260nm.
True
This is why the 260/280 ratio is important for assessing purity.
What is the dilution factor used in the described procedure?
1 in 100
This dilution factor is standard for preparing samples for spectrophotometry.
What does an A260 value of 820.5 ng/ul suggest about the sample?
The concentration of the DNA sample
This value is derived from the absorbance reading.
What wavelength range is typically used for DNA and protein absorbance measurements?
220nm to 340nm
This range covers the relevant absorbance for nucleic acids and proteins.
What is the absorption value of a 50ug/ml pure DNA solution at 260nm?
1
The absorption value indicates the concentration of DNA in the solution.
What is the 260nm:280nm ratio for a pure DNA solution?
Greater than 1.7
This ratio is used to assess the purity of DNA.
What is the absorption value of a 40ug/ml pure RNA solution at 260nm?
1
Similar to DNA, this value indicates the concentration of RNA.
What is the 260nm:280nm ratio for a pure RNA solution?
Greater than 1.8
This ratio indicates the purity of RNA.
How do you determine the concentration of genomic DNA preparation?
A260 X 50 X 100
This formula calculates the concentration in ug/ml.
What is the dilution factor used in determining the concentration of genomic DNA preparation?
1 in 100
This refers to the dilution of the DNA sample before measurement.
How do you convert concentration from ug/ml to ug/ul?
Divide by 1000
This conversion is necessary for appropriate volume measurements.
What percentage of agarose is used for electrophoresis of genomic DNA?
0.8%
This concentration is suitable for estimating the integrity and concentration of DNA.
What is the first step in preparing a gel template for electrophoresis?
Seal the ends of the template with masking tape
This prevents the gel mixture from leaking.
What should be added to the gel mixture to achieve the final agarose concentration?
Ethidium bromide
This is used for visualizing DNA under UV light.
What is the final concentration of ethidium bromide added to the agarose gel?
0.5ug/ml
This concentration is effective for DNA visualization.
How long should the gel mixture be allowed to set?
Approximately 20 minutes
This allows the agarose to solidify properly.
What components are combined to prepare the DNA sample for loading into the gel?
1ug of DNA, 2ul of 6X loading buffer, and water to 12ul
This mixture ensures proper loading and visualization of DNA.
At what voltage should the agarose gel be electrophoresed?
100V
This voltage is standard for most DNA electrophoresis protocols.
For how long should the gel be electrophoresed?
Approximately 30 minutes
This duration allows sufficient separation of DNA fragments.
What equipment is used to examine the gel after electrophoresis?
UV transilluminator
This device allows for the visualization of DNA stained with ethidium bromide.
