Laboratory 5 Flashcards
What are you doing in this experiment?
Extraction of total RNA from peripheral blood leucocytes
What do we use to lyse our wbcs?
Lysed by guanidine thiocyanate in the presence of acid phenol
How do we get our RNA after lysing our wbcs?
After phase separation the DNA partitions into the organic phase and the RNA remains in the aqueous phase
How do we get DNA from this experiment?
DNA is recovered from the organic phase by back extraction with Tris base
How do you extract the RNA?
(5)
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
What does red blood cell lysis solution?
(3)
155mM ammonium chloride
10mM potassium hydrogen carbonate
1mM EDTA
What do you do with the aqueous phase?
(6)
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
What reagents do we need to split the whole blood into an aqueous and an organic phase?
(3)
Red blood cell lysis solution
Tri reagent
Bromochloropropane
What reagents do we need to purify our RNA?
(3)
Isopropanol
75% ethanol
RNase free water
How do we purify our DNA?
(9)
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
What reagents are needed to purify DNA?
(4)
Back extraction buffer
Isopropanol
70% ethanol then centrifuge
TE
When purifying DNA, what does the organic phase contain?
Organic phase contains protein
At what absorbance does DNA absorb UV light?
260nm
At what absorbance does protein absorb UV light?
280 nm
What is the 260:280nm ration used for?
To determine the concentration and purity of DNA
