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2.1 Cell and Molecular Biology > Laboratory 5 > Flashcards

Laboratory 5 Flashcards

1
Q

What are you doing in this experiment?

A

Extraction of total RNA from peripheral blood leucocytes

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2
Q

What do we use to lyse our wbcs?

A

Lysed by guanidine thiocyanate in the presence of acid phenol

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3
Q

How do we get our RNA after lysing our wbcs?

A

After phase separation the DNA partitions into the organic phase and the RNA remains in the aqueous phase

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4
Q

How do we get DNA from this experiment?

A

DNA is recovered from the organic phase by back extraction with Tris base

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5
Q

How do you extract the RNA?
(5)

A

Mix whole blood and red blood cell lysis solution and incubate at room temp

Centrifuge and pipette of supernatant

Resuspend and add Tri reagent then incubate

Add bromochloropropane, vortex, incubate then centrifuge

Split into aqueous phase and organic phase

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6
Q

What does red blood cell lysis solution?
(3)

A

155mM ammonium chloride
10mM potassium hydrogen carbonate
1mM EDTA

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7
Q

What do you do with the aqueous phase?
(6)

A

Transfer aqueous phase to a fresh tube and add equal vol of isopropanol, retain organic phase

Vortex and incubate then pellet RNA in centrifuge

Remove supernatant and add 1ml of 75% ethanol, vortex, centrifuge

Pipette off ethanol and air dry

Resuspend in 25ul RNase free water and heat to 65 degrees

Estimate RNA concentration in a UV spec

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8
Q

What reagents do we need to split the whole blood into an aqueous and an organic phase?
(3)

A

Red blood cell lysis solution

Tri reagent

Bromochloropropane

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9
Q

What reagents do we need to purify our RNA?
(3)

A

Isopropanol

75% ethanol

RNase free water

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10
Q

How do we purify our DNA?
(9)

A

Add back extraction buffer, vortex and retain for 10 mins

Centrifuge

Transfer aqueous phase to fresh tube and add isopropanol

Organic phase contains protein

Vortex and incubate then centrifuge

Aspirate supernatant and wash pellet with 70% ethanol then centrifuge

Aspirate supernatant and dry pellet

Resuspend DNA in TE and incubate

Estimate DNA concentration in a UV spec

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11
Q

What reagents are needed to purify DNA?
(4)

A

Back extraction buffer

Isopropanol

70% ethanol then centrifuge

TE

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12
Q

When purifying DNA, what does the organic phase contain?

A

Organic phase contains protein

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13
Q

At what absorbance does DNA absorb UV light?

A

260nm

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14
Q

At what absorbance does protein absorb UV light?

A

280 nm

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15
Q

What is the 260:280nm ration used for?

A

To determine the concentration and purity of DNA

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16
Q

How do we use the nanodrop?
(4)

A

Blank the nanodrop using water

Remove water with tissue

Add 2ul of DNA solution

Read absorbance at 260nm and 280 nm

17
Q

What should your ration be greater than?

A

1.7

2.1 Cell and Molecular Biology (26 decks)

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