Flowchart of dna blot hybridization analysis



Download 16.29 Kb.
Date conversion04.02.2017
Size16.29 Kb.

HC70AL SPRING 2004 PROFESSOR BOB GOLDBERG

FLOWCHART OF DNA BLOT HYBRIDIZATION ANALYSIS

Preparing the agarose gel with a 40-tooth comb




Preparing of 31 samples to be loaded on the gel




Loading 31 samples + 2 lanes (for 1-kb ladder) on the agarose gel




Running the gel for 1.5-2 hours




Taking a picture of the gel


Treating the gel with Depurination solution [0.25 M Hydrochloric acid (HCl)]

for 10 minutes with gentle shaking (~50 rpm)




Rinsing the gel with water (TWICE) to remove HCl solution




Treating the gel with ~200-250 mL of Denaturation solution

for 15 minutes with gentle shaking (~50 rpm)


Pouring off the denaturation solution




Treating the gel with another ~200-250 mL of Denaturation solution

for 15 minutes with gentle shaking (~50 rpm) AGAIN
Rinsing the gel with water ONCE to remove Denaturation solution
Treating the gel with ~200-250 mL of Neutralization solution

for 15 minutes with gentle shaking (~50 rpm)




Pouring off the neutralization solution




Treating the gel with another ~200-250 mL of Neutralization solution

for 15 minutes with gentle shaking (~50 rpm) AGAIN



Blotting the gel for at least 16 hours

(same as the first step on next page)


Blotting the gel for at least 16 hours





Disassembling the blotting apparatus

Marking the wells on the Nylon membrane (the blot)

Treating the blot with a UV source for 5 minutes

to crosslink nucleic acids to the membrane

Washing the blot in a 6X SSC solution



Baking the blot at 80oC in a vacuum oven for 1-1.5 hours

(If the blot is NOT hybridized immediately and stored for at least a few days)

Storing the blot in a Seal-A-Meal bag until needed



Preparing pre-hybridization and hybridization solutions



Prehybridizing the blot in the prehybridization solution

at 42oC for at least 1-2 hours.

Preparing radioactively labeled DNA probes using

Random Primer Labeling Kit

Hybridizing the blot in the hybridization solution

containing denatured DNA probes at 42oC for at least 16 hours.

Preparing two wash (low and high stringency) solutions



Removing the blot from the hybridization bag

(same as the first step on next page)


Removing the blot from the hybridization bag



Washing the blot in the low stringency solution at room temperature

for 15 minutes with gentle shaking (~50 rpm)

Repeating the wash with the low stringency solution

for another 15 minutes with gentle shaking (~50 rpm)

Washing the blot in the high stringency solution at 60oC

for 30 minutes with gentle shaking (~50 rpm)

Repeating the wash with the high stringency solution

for another 15 minutes with gentle shaking (~50 rpm)

Blotting the blots on several layers of Kimwipes to remove excess liquid



Placing the blots on a piece of 3MM Whatman paper wrapped

with plastic wrap

Estimating the amount of probes (in cpm) on the blot using a handheld

Geiger counter

Setting up the blot exposing to X-ray film in the darkroom



Exposing the blot to X-ray film at -80oC or room temperature

for as short as 15 minutes to several hours or for one day

(Depending on the probe count)




Developing the exposed X-ray film (autoradiogram)






Marking well positions and labeling samples on the autoradiogram



Repeating exposure (if necessary)







The database is protected by copyright ©dentisty.org 2016
send message

    Main page