Biotechnology Engineering

1. Recovery of DNA
from an Agarose Gel
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Lecture- 16

2. Methods for Elution of DNA
from an Agarose Gel
• Organic extraction
• Electroelution into dialysis bags
• Enzymatic digestion with agarase
• Optimized freeze sqeeze method
• Electrophoresis onto DEAE-cellulose membrane
• Affinity chromatography
• Crush and soak method
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3. Organic Extraction
Materials
• Water bath set to 67°C
• Microcentrifuge tubes (≥1 ml)
• Ice bucket and ice
• Vortex mixer
• Microcentrifuge
• Scalpel or razor blade
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4. Reagents
• Low melting temperature agarose
• TE Buffer warmed to 67°C
• Buffer-equilibrated phenol
• Chloroform
• Phenol/chloroform (1:1)
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5. Procedure
1. Electrophorese DNA in a low melting temperature (LMT) agarose gel
prepared in 1X TAE Buffer.
2. Set a water bath for 67°C.
3. Prewarm the TE at 67°C.
4. Excise the gel fragment containing the DNA.
5. Place the DNA slice in a microcentrifuge tube.
6. Estimate the volume of the slice. If the slice is significantly greater
than 200 mg, break the agarose slice into smaller pieces and place each
agarose piece in a separate microcentrifuge tube.
7. Melt the gel slice at 67°C for 10 minutes.
8. Add the appropriate volume of TE buffer (prewarmed to 67°C) so that
the final concentration of agarose is 0.5%.
9. Maintain the samples at 67°C until you are ready to phenol extract.
10. Add an equal volume of buffer-equilibrated phenol. 5

6. 11. All subsequent steps can be done at room temperature.
12. Vortex for 15 seconds.
13. Centrifuge at maximum speed in a microcentrifuge for 3 minutes.
14. Carefully remove the top aqueous phase. The interface of white
debris is the agarose, which can contain some trapped DNA. This can be
back-extracted with TE to maximize yield.
15. Place aqueous phase in a clean microcentrifuge tube.
16. Repeat steps 10 - 15.
17. Add an equal volume of phenol/chloroform to the aqueous phase.
18. Vortex for 15 seconds.
19. Centrifuge at maximum speed in a microcentrifuge for 3 minutes.
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7. 20. Remove the aqueous phase and place in a clean
microcentrifuge tube.
21. Repeat the extraction with an equal volume of chloroform.
22. Remove the aqueous phase and place in a clean
microcentrifuge tube.
23. Chill the aqueous phase for 15 minutes on ice.
24. Centrifuge in a microcentrifuge at high speed for 15 minutes
at 4°C.
25. Carefully decant the supernatant into a clean microcentrifuge
tube.
26. Ethanol precipitate the DNA in the supernatant following
standard protocols.
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8. Electroelution
• Electroelution is a reliable and consistent recovery method.
• For small fragments, a typical yield would be 50 - 85%, but as
fragment length increases, yield can drop as low as 20%.
• It requires transfer of individual gel slices into dialysis bags
• This method is time consuming, requires more vigilance and is
tedious to perform
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9. Method
• Transfer the agarose gel slice to a dialysis bag sealed from one
end and filled with 0.25X TBE
• Pass electric current through the bag (4 - 5 V/cm for 2 -
3hours). This process can be monitored with a hand-held,
long-wavelength UV lamp.
• Reverse the polarity of the current for 1 minute. This will
remove the DNA from the wall of the bag.
• Transfer all of the buffer surrounding the gel slice to a plastic
tube.
• Wash out the bag with a small amount of 1X TAE Buffer.
• Transfer the solution to a plastic tube.
• Purify the DNA from 1X TAE solution using phenol/chloroform
extractions. 9

10. Enzymatic Digestion
• Digest the polysaccharide backbone of agarose to alcohol-
soluble oligosaccharides with beta-agarase enzyme.
• DNA from a low melting temperature agarose gel can be
recovered after the gel is melted and digested with the beta-
agarase enzyme.
• This method is particularly useful for recovering large DNA
(>10 kb)
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11. Method
• Equilibrate the gel slice with 10 volumes of 1X Beta-agarase
buffer for 60 minutes at room temperature
• Melt the gel slice at 70°C (approximately 15 minutes) and cool
up to 450C.
• Add 1 unit of beta-agarase for 200 mg (approximately 200 μl)
of 1% agarose gel
• Place the solution at -20°C for 15 minutes. Centrifuge the
solution for 15 minutes at 4°C.
• Extract DNA
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12. Freeze Squeeze Method
• Gel slice is kept in an Eppendorf tube and elution buffer is
added up to the level of gel slice
• The tube is heated at 65° C for 5 min
• Freeze at -70°C for 5 to 15 min
• Centrifuge for 15 min at room temperature
• Remove the supernatant which contains the DNA
• Place the supernatant in a clean microcentrifuge tube
• Do a phenol/chloroform extraction
• Follow with a chloroform extraction.
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13. Advantages
• Simple and fast to perform
• 80-95% of small fragments are easily eluted out
• DNA retains shape after precipitation
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14. Electrophoresis onto DEAE-
Cellulose Membrane
• At low concentration of salts, DNA binds avidly to DEAE
cellulose membrane
• A slit, just in front of the DNA band, is made in the agarose gel
• DEAE cellulose (paper form) is then inserted in the slit so that
DNA can bind to it
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