TITLE: Quick & Dirty DNA Extraction
AUTHORS: Sam Mantel, Andrea Sweigart

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Modified from Cheung et al., 1993 </div></div>

[STEPS]
?. [Prep]
Pre-heat Quick & Dirty extraction buffer with detergent in a  incubator.
60 °C
?. [Prep]
Place one steel ball bearing into each sample tube.
?. [Extract and Wash DNA]
When tissue is ground to a fine powder centrifuge briefly to remove powder from the inside of caps.
?. [Make Quick & Dirty extraction buffer with detergent (500mL)]
Add roughly   nanopure H2O to a  glass bottle with a stir bar and stir.
200 ml
1 L
?. [Extract and Wash DNA]
Snap bottom back on to plate and shake in tissue homogenizer for  on highest seed.    Note: Shake plate without lid so that casing of the homogenizer holds caps in place.    Distribute samples evenly across plate so that samples are ground evenly.
?. [Extract and Wash DNA]
Add  pre-heated Q&D extraction buffer with detergent to each sample.
350 µl
?. [Extract and Wash DNA]
Incubate samples at  for  to  .Note: To prevent caps from popping off while incubating, place inverted plate bottom over caps and hold down with a weight.
60 °C
?. [Extract and Wash DNA]
Centrifuge   to pellet tissue.
Centrifuge: 3700 33, 20 min, 22 10
?. [Make Quick & Dirty extraction buffer with detergent (500mL)]
Return to  bottle and autoclave for  .
1 L
?. [Make Quick & Dirty extraction buffer with detergent (500mL)]
Gently add  5% sarkosyl solution to Q&D buffer.    Note: Sarkosyl solution is a a detergent and will foam if poured to quickly.
100 ml
?. [Extract and Wash DNA]
Near the end of the spin, transfer   per sample ice cold isopropanol to a new plate.
200 µl
?. [Make Quick & Dirty extraction buffer with detergent (500mL)]
Transfer  Tris base to bottle.
80 ml
?. [Make Quick & Dirty extraction buffer with detergent (500mL)]
Transfer  EDTA to bottle.
56 ml
?. [Make Quick & Dirty extraction buffer with detergent (500mL)]
Transfer  NaCl to bottle.
46.7 g
?. [Make Quick & Dirty extraction buffer with detergent (500mL)]
Transfer  sodium metabisulfite to bottle.
3 g
?. [Make Quick & Dirty extraction buffer with detergent (500mL)]
Stir until dissolved.
?. [Make Quick & Dirty extraction buffer with detergent (500mL)]
Transfer solution to graduated cylinder and add nanopure H2O to bring volume to  .
400 ml
?. [Make Quick & Dirty extraction buffer with detergent (500mL)]
Allow solution to cool to room temperature.
?. [Make Quick & Dirty extraction buffer with detergent (500mL)]
Swirl solution gently to mix.    Note: Once detergent has been added, do not autoclave, detergent will degrade at high heat.
?. [Prep]
Remove plate from -80 and quickly remove bottom from plate and float in liquid nitrogen to keep tissue frozen.
?. [Extract and Wash DNA]
Open tubes and discard caps.
?. [Extract and Wash DNA]
Cover with clean caps and invert gently to mix.
?. [Extract and Wash DNA]
Add  ammonium acetate to each sample.
150 µl
?. [Extract and Wash DNA]
Invert samples gently every  .Note: Caps will likely be loose, close tubes completely before inverting.
?. [Prep]
Place isopropanol, 70% ethanol, and 95% ethanol in the freezer to chill.
?. [Extract and Wash DNA]
Transfer  , or as much as you can, of supernatant from each sample to isopropanol plate.
300 µl
?. [Extract and Wash DNA]
Recap tubes and invert several times to mix.
?. [Extract and Wash DNA]
Incubate at room temperature for  to
?. [Extract and Wash DNA]
Centrifuge   to pellet DNA.
Centrifuge: 3700 33, 20 min, 4 10
?. [Extract and Wash DNA]
Slowly pour off supernatant without disturbing pelleted DNA.
?. [Extract and Wash DNA]
Add  ice cold 70% ethanol to each sample.
500 µl
?. [Extract and Wash DNA]
Cover with clean caps and invert gently to mix.
?. [Extract and Wash DNA]
Centrifuge   to wash pellet.
Centrifuge: 3700 33, 10 min, 4 10
?. [Extract and Wash DNA]
Slowly pour off supernatant without disturbing pelleted DNA.
?. [Extract and Wash DNA]
Add  ice cold 95% ethanol to each sample.
500 µl
?. [Extract and Wash DNA]
Cover with clean caps and invert gently to mix.
?. [Extract and Wash DNA]
Centrifuge   to wash pellet.
Centrifuge: 3700 33, 5 min, 4 10
?. [Extract and Wash DNA]
Slowly pour off supernatant without disturbing pelleted DNA.
?. [Dry and Re-suspend DNA]
Place the flat on a paper towel and cover the open tubes with a KimWipe.
?. [Dry and Re-suspend DNA]
Allow pellets to dry overnight.
?. [Dry and Re-suspend DNA]
Once pellet is dry, and no residual liquid remains in the tubes, re-suspend pellets with  -  of TE buffer or nanopure H2O depending on pellet size, desired concentration, and future uses.
20 µl
100 µl