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4.1 Molecular Biology > Lab 2: Extraction Of RNA, DNA and Protein > Flashcards

Lab 2: Extraction Of RNA, DNA and Protein Flashcards

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1
Q

What method is used for RNA isolation from peripheral blood leucocytes?

A

Acid phenol guanidine thiocyanate method

This method lyses white blood cells using guanidine thiocyanate in the presence of acid phenol.

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2
Q

What happens to DNA and RNA during the phase separation in RNA extraction?

A

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.

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3
Q

What is the first step in the extraction of total RNA from peripheral blood leucocytes?

A

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.

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4
Q

What is the incubation time for the blood and lysis solution mixture?

A

10 minutes

The mixture is incubated at room temperature and inverted occasionally.

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5
Q

What is the purpose of centrifuging the sample after incubation with the lysis solution?

A

To form a white pellet of unlysed leucocytes

The supernatant contains lysed erythrocytes.

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6
Q

What reagent is added to the resuspended leucocyte pellet after centrifugation?

A

500ul of Tri reagent

Tri reagent facilitates RNA extraction by lysing the cells.

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7
Q

What is added to the cell suspension after the Tri reagent, and what is its purpose?

A

50ul of bromochloropropane; to aid in phase separation

This step is crucial for effective extraction of RNA.

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8
Q

At what temperature should the centrifugation occur after adding bromochloropropane?

A

4°C

This temperature helps to stabilize the RNA during the extraction process.

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9
Q

What is done with the upper (aqueous) phase after centrifugation?

A

Transfer to a fresh tube and add an equal volume of isopropanol

This step precipitates the RNA from the aqueous phase.

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10
Q

What is the purpose of adding 1ml of 75% ethanol after RNA pellet formation?

A

To wash the RNA pellet

This step helps to remove impurities and residual salts.

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11
Q

What is the final step in resuspending the RNA?

A

Resuspend in 25ul of RNase free water and heat to 65°C

Heating helps to dissolve the RNA effectively.

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12
Q

How is RNA concentration estimated after resuspension?

A

Using a UV spectrophotometer

This method provides a quantifiable measure of RNA concentration.

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13
Q

What is the purpose of adjusting the pH of the solution in DNA extraction?

A

To allow DNA to partition to the aqueous phase

This is typically done with 1M Tris base.

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14
Q

What components are included in the back extraction buffer for DNA extraction?

A
  • 4M guanidine thiocyanate
  • 50mM sodium citrate
  • 1M Tris

These components help in the extraction process of DNA.

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15
Q

What is the first step after adding back extraction buffer to the retained organic phase?

A

Vortex and retain for 10 minutes at room temperature

This step ensures thorough mixing of the buffer with the organic phase.

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16
Q

What is the centrifugation time and temperature for the DNA extraction process?

A

5 minutes at full speed in a benchtop microfuge at 4°C

This step separates the aqueous phase from the organic phase.

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17
Q

What is added to the aqueous phase after centrifugation in DNA extraction?

A

300ul isopropanol

This aids in precipitating the DNA.

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18
Q

What should be done with the organic phase after transferring the aqueous phase?

A

Retain it for Protein extraction

The retained organic phase contains proteins that can be extracted.

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19
Q

How is the DNA pellet washed after centrifugation?

A

With 70% ethanol

This wash step helps to purify the DNA pellet.

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20
Q

What is the final step in resuspending the DNA pellet?

A

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.

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21
Q

What is the method used to estimate DNA concentration?

A

UV spectrophotometry

This technique allows for quantification of DNA concentration.

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22
Q

What is added to the retained organic phase for protein extraction?

A

750ul of isopropanol

This is the first step in the protein extraction process.

23
Q

What is the washing solution used for protein pellet washing?

A

0.3M guanidine hydrochloride in 95% ethanol

This solution is used to clean the protein pellet.

24
Q

What temperature and duration are required for boiling the protein sample loading buffer?

A

100°C for 5 minutes

This step prepares the protein for analysis.

25
Q

At what wavelength does DNA absorb UV light?

A

260nm

This absorption is used to determine DNA concentration.

26
Q

At what wavelength do proteins absorb UV light?

A

280nm

This is used to assess the purity of DNA solutions.

27
Q

What is the significance of the ratio between the absorption at 260nm and 280nm?

A

It indicates both the concentration and purity of DNA

A pure DNA solution should have a 260/280 ratio greater than 1.7.

28
Q

What should be done before measuring DNA concentration with a nanodrop?

A

Blank the nanodrop using water

This ensures accurate measurements.

29
Q

What is the procedure for measuring DNA concentration using a nanodrop?

A

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.

30
Q

What does an A260 value of 1 indicate in terms of DNA concentration?

A

50ug/ml

This is a standard conversion used in DNA quantification.

31
Q

How is the test DNA concentration determined using the A260 reading?

A

A260 X 50 X 100 = concentration in ug/ml

This formula helps convert absorbance to concentration.

32
Q

What is the typical storage temperature for DNA samples?

A

-80°C

This is recommended to preserve DNA integrity.

33
Q

What is the expected A260 and A280 reading for a 50.00 ng/ul DNA solution?

A

A260: 16.411, A280: 8.861

These values are indicative of the DNA concentration and purity.

34
Q

Fill in the blank: A pure DNA solution should have a 260/280 ratio greater than ______.

A

1.7

This ratio is crucial for determining DNA purity.

35
Q

True or False: Proteins can interfere with the measurement of DNA concentration at 260nm.

A

True

This is why the 260/280 ratio is important for assessing purity.

36
Q

What is the dilution factor used in the described procedure?

A

1 in 100

This dilution factor is standard for preparing samples for spectrophotometry.

37
Q

What does an A260 value of 820.5 ng/ul suggest about the sample?

A

The concentration of the DNA sample

This value is derived from the absorbance reading.

38
Q

What wavelength range is typically used for DNA and protein absorbance measurements?

A

220nm to 340nm

This range covers the relevant absorbance for nucleic acids and proteins.

39
Q

What is the absorption value of a 50ug/ml pure DNA solution at 260nm?

A

1

The absorption value indicates the concentration of DNA in the solution.

40
Q

What is the 260nm:280nm ratio for a pure DNA solution?

A

Greater than 1.7

This ratio is used to assess the purity of DNA.

41
Q

What is the absorption value of a 40ug/ml pure RNA solution at 260nm?

A

1

Similar to DNA, this value indicates the concentration of RNA.

42
Q

What is the 260nm:280nm ratio for a pure RNA solution?

A

Greater than 1.8

This ratio indicates the purity of RNA.

43
Q

How do you determine the concentration of genomic DNA preparation?

A

A260 X 50 X 100

This formula calculates the concentration in ug/ml.

44
Q

What is the dilution factor used in determining the concentration of genomic DNA preparation?

A

1 in 100

This refers to the dilution of the DNA sample before measurement.

45
Q

How do you convert concentration from ug/ml to ug/ul?

A

Divide by 1000

This conversion is necessary for appropriate volume measurements.

46
Q

What percentage of agarose is used for electrophoresis of genomic DNA?

A

0.8%

This concentration is suitable for estimating the integrity and concentration of DNA.

47
Q

What is the first step in preparing a gel template for electrophoresis?

A

Seal the ends of the template with masking tape

This prevents the gel mixture from leaking.

48
Q

What should be added to the gel mixture to achieve the final agarose concentration?

A

Ethidium bromide

This is used for visualizing DNA under UV light.

49
Q

What is the final concentration of ethidium bromide added to the agarose gel?

A

0.5ug/ml

This concentration is effective for DNA visualization.

50
Q

How long should the gel mixture be allowed to set?

A

Approximately 20 minutes

This allows the agarose to solidify properly.

51
Q

What components are combined to prepare the DNA sample for loading into the gel?

A

1ug of DNA, 2ul of 6X loading buffer, and water to 12ul

This mixture ensures proper loading and visualization of DNA.

52
Q

At what voltage should the agarose gel be electrophoresed?

A

100V

This voltage is standard for most DNA electrophoresis protocols.

53
Q

For how long should the gel be electrophoresed?

A

Approximately 30 minutes

This duration allows sufficient separation of DNA fragments.

54
Q

What equipment is used to examine the gel after electrophoresis?

A

UV transilluminator

This device allows for the visualization of DNA stained with ethidium bromide.

4.1 Molecular Biology (14 decks)

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