Plant Lab Protocols / Methodology for Nucleic Acids
Extraction of lambda (l) DNA
Bacteriophage DNA is required for many experiments in molecular biology. Its restriction fragments serve as size markers. Phage DNA is used in certain recombinant DNA experiments as well. A method to separate the DNA from purified phage particles is described below. See previous pages for harvesting of purified phage particles.
Principle
Proteinase K is used to digest the viral protein coat and the DNA is precipitated with acetate and alcohol.
Materials
» Dialysis Bag
» Dialysis Buffer
50mM Tris-HCl (pH 8.0)
10mM NaCl
10mM MgCl2
» EDTA (0.2M) sodium salt
» Pronase or proteinase K
» SDS (20% solution)
» Phenol
» Chloroform
» 3M Sodium acetate (pH 5.5)
» Absolute alcohol
» 70%ethanol
» TE buffer
10mMTris-HCl (pH7.8)
1mMEDTA
» Phenol (freshly redistilled phenol equilibrated with TE Buffer)
(Mix equal volumes of phenol and TE buffer, stand for a few hours and drain off excess aqueous upper phase.)
Procedure
1.
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Transfer the purified phage particles into a dialysis bag and dialyse against a 1000-fold volume of dialysis buffer for 1h.
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2.
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Change the dialysis buffer and dialyse for another 1h.
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3.
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Now, carefully transfer the content of the dialysis bag into a centrifuge tube.
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4.
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Add EDTA to give a final concentration of 20mM.
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5.
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Add proteinase K to a final concentration of 50g/mL or protease to 0.5mg/mL.
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6.
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Add SDS to a final concentration of 0.5%. Mix thoroughly by inverting the tube several times.
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7.
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If proteinase K is used, incubate for 1h at 65°C (for pronase 1h at 37°C).
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8.
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Add an equal volume of phenol equilibrated with TE buffer. Mix by inverting the tubes several times. Centrifuge at 1600 x g for 5 min at room temperature. Transfer the aqueous phase to a clean tube.
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9.
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Extract the aqueous phase once again with 50 : 50 mixture of equilibrated phenol and chloroform.
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10.
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Extract the aqueous phase again with an equal volume of chloroform.
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11.
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Add one tenth volume of 3M sodium acetate, equal volume of absolute alcohol, mix thoroughly and stand at -20°C for a few hours. Centrifuge and wash the pellet with 70% ethanol. Redissolve the DNA in a small volume of TE buffer.
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12.
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Determine the DNA concentration spectrophotometrically. Store the DNA solution in small aliquots at -20°C.
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