Various methods of extracting DNA from the blood samples collected on Caamaño have been tested.  These range from phenol-chloroform extraction to salting out to simple extraction (DNA precipitation) from the preservative without further digestion by [proteinase K].  Additionally, multiple methods of preparing samples for PCR have been tested.  These range from use of DNA extracted through the methods mentioned above to boiling the preserved blood sample to microwaving the preserved blood sample prior to the PCR reaction.

Results of the methods are listed and described below:

• Phenol-Chloroform extraction
• Salting Out
• Digestion-free extraction
• Boiling
• Microwaving
• QIAGEN (QIAamp tissue extraction kit)

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• Use 0.01-0.50 g or fresh tissue (liver works very well) and place in a 1.5 ml microcentrifuge tube.
• Add 200-400 ul lysis buffer and macerate tissue.  Add more lysis buffer if sample is too viscous.
• Microfuge for 3 min. at 10,000 rpm.
• Remove supernatant and place in a new microfuge tube.
• Add equal volume of buffer-equilibrated phenol and invert gently.
• Microfuge for 10 min. at 10,000 rpm.
• Remove upper aqueous layer with a pipetter and place in a new microfuge tube.
• Add equal volume of phenol:chloroform (1:1) and invert gently.
• Microfuge for 5 min. at 10,000 rpm.
• Remove upper aqueous layer with a pipetter and place in a new microfuge tube.
• Add equal volume of chloroform and invert gently.
• Microfuge for 3 min. at 10,000 rpm.
• Remove upper aqueous layer with a pipetter and place in a new microfuge tube.
• Add 1/2 volume 7.5 M NH₄OAc.
• To the new volume add enough ethanol (100% / 200 proof) to make the solution 66% ethanol and invert gently.  DNA precipitate should appear.  Wait as long as 10 min. for all samples to develop precipiate.
• Microfuge for 10 min. at 10,000 rpm.
• Carefully decant liquid so not to dislodge pellet.
• Finger flick to dislodge pellet and then fill each tube with 70% ethanol.  Let stand for 10 min.
• Microfuge for 10 min. at 10,000 rpm.
• Decant off liquid and use a Kimwipe to swab dry remaining fluid.  Don't touch the pellet!
• Air dry pellet.
• Add 100 ul sterile TE to each sample, disolve DNA, and freeze at -20^oC until needed.

This picture shows some DNA extracted by the phenol-chloroform method (the group of 4 on the right side of the gel).  These results should not be considered typical.  I have reason to believe that the blood samples have been degraded and as a result the DNA is sheared into smaller pieces (which creates the "smear" effect).  Notice: that the DNA extracted from the later females was much longer, with the majority (though sheared) located nearer the top of the gel.

• Place 3 ml nuclei lysis buffer in a 15 ml polypropylene centrifuge tube.
• Put blood sample into tube with nuclei lysis buffer and resuspend.
• Add 0.2 ml of 10% SDS and 0.5 ml protease K solution and allow to digest at 37^oC overnight.
• Add 1 ml saturated NaCl and shake vigorously for 15 sec.
• Centrifuge at 2500 rpm for 15 min.
• Transfer supernatant (containing DNA) to another 15 ml polypropylene tube.
• Add 2 volumes of ice cold absolute (100% / 200 proof) ethanol and gently invert several times (until DNA precipitates).
• Remove precipitated DNA strands with a plastic spatula or pipette and place in a 1.5 ml microfuge tube that contains 100-200 ul TE buffer.
• Allow DNA to dissolve for 2 hrs. at 37^oC.
• Quantify DNA.

The standard salting out procedure was followed for the group of samples (4) located on the left side of the gel.  The "smeared" effect is likely due to some degeneration of the blood samples prior to extraction of the DNA.  With high quality blood or tissue samples the standard salting out method works well.
 

Above are seen the effects of various digestion (by proteinase K) time periods on the "salting out" DNA extraction process.  These digestion times are very long.  The recommended time ranges between 2-3 hours and overnight.  Digestion time and efficiency is affected by the incubation temperature during digestion as well.  Temperatures used range from room temperature up to 55-57^o C (which is the optimum temperature for the activity of the enzyme, proteinase K).

This was a shortened form of the salting out procedure.  Specifically, the overnight digestion step was omitted (so no 10% SDS or protease K solution was used).  Blood samples were simply resuspended in nuclei lysis buffer and extracted by salting out as outlined above.

Perhaps you are getting tired of this picture.  :)    The digestion-free samples (5) are located in the center of the gel.  These samples show substantial "shearing & smearing."  Degredation of the blood samples has likely taken place prior to DNA extraction.  Because of blood sample storage conditions (basically in a lysis/digestion buffer solution) DNA extraction appears to work just as well without further digestion of the blood samples by proteinase K.

1. This was a modification of digestion-free salting out procedure.  The only difference being that after the blood samples were resuspended in the nuclei lysis buffer they were heated to 100^oC for 15 min.  (Other temperatures were tested as well.)

Results of extractions made at different temperatures are shown above. The extractions at the 4 temperatures were made on the same 3 individuals (X, Y, Z).  There does not seem to be any indication that a higher temperature helps in the extraction of the DNA.  (???)
 

2. Alternatively, another boiling procedure was tested where digestion with proteinase K preceded the boiling:

• Placed app. 20 ul of blood sample in a microfuge tube and added 1 ul of 20 mg/ml proteinase K solution
• Incubated at 55^oC for 15 min.
• Vortexed well and incubated at 55^oC for another 15 min.
• Added 179 ul distilled water to bring volume to 200 ul.
• Heated in boiling water for 5 min.
• Cooled on ice
• Used 1 ul directly for PCR (so no actual DNA extraction took place)

The 3 center lanes (with a suffix of "B") on the gel show the results of PCR using the blood sample digested and boiled just prior to running the PCR program.  This seems to be a useful and fast method when compared with the results of the other (QIAGEN & microwaving) methods, at least when performed just prior to use in the PCR reaction.  (See below . . .)  The actual band(s) of interest are those just below the 298 base pair (bp) marker of the 1 Kb molecular weight ladder.
 

A second (later) use, after storage at 4^o C, of the prepared (boiled) DNA template for the PCR reaction does not appear to work well.  This is likely due to degredation of the DNA by enzymes present in the solution.  A benefit of DNA extraction methods is that the DNA is separated from enzymes found within the cell that degrade DNA.  This allows extracted samples to be stored and used later.  However, this method seems to be a one-time use one.  (I have not tried using stored sample products produced by this method again, so I cannot be positive of this.)

This was a treatment in preparation for direct PCR of blood samples (so no actual DNA extraction took place):

• Put 1 ul blood sample into microfuge tube and microwave on high for 1 min.
• Cooled on ice
• Added PCR master mix (containing water, buffer, MgCl₂, dNTPs, primers, and Taq) and ran PCR

The rightmost 3 lanes on this gel (with a suffix of "M") contain the product of the PCR reaction completed on the microwaved samples.  It does not appear to work very well here.  There is no amplification of specific bands.  Only a smear of DNA is visible.

QIAGEN's tissue extraction kit was used according to the included instructions.  Their solutions are not labeled as to their contents.  Microfuge tubes are used and a DNA containing solution with a volume from 100-400 ul is obtained.  A general schematic of the procedure may be seen here (diagram).

Lysis Buffer:
    100 mM EDTA
    10 mM Tris (pH 7.5)
    1% SDS

TE:
    10 mM Tris-HCl
    0.2 mM Na2EDTA
    adjust pH to 7.5

Nuclei Lysis Buffer:
    10 mM Tris-HCl
    400 mM NaCl
    2 mM Na₂EDTA
    adjust pH to 8.2

Protease K Solution:
    1 mg/ml protease K
    1% SDS
    2 mM Na2EDTA
        *** Store at -20^oC
 
 

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