Mimulus:DNA Extraction Protocol

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Here is the protocol that is used in the Willis lab:

Mimulus Corolla (Leaf) DNA Prep:

Things to do first:

1)	Turn on water bath (60 degrees C).

2)	Get liquid nitrogen.

3)	Get sample tube/plates from -80 degree freezer. Keep samples chilled on ice.

4)	Add one bead to each tube.

DO IN HOOD (except for freezing, grinding, and spinning steps)

1)	Make Extraction Master Mix: Measure out 500 uL of CTAB extraction buffer per sample into trough.  Add 1 uL  B-mercaptoethanol per 500 uL of CTAB extraction buffer to same trough.  This “master mix” should include: [(# of samples + 10%) x 500 uL of CTAB] + [(# of samples + 1) x 1 uL B-mercaptoethanol].
 * For one 96 well plate: 		52mL buffer + 104 uL B-mercaptoethanol
 * For two 96 well plates: 	104mL buffer + 208 uL B-mercaptoethanol

2)	Remove bottom of tube rack. Dip tubes/plates in liquid nitrogen (for up to 10 seconds).  Clamp plates into shaker rack in Genogrinder.  ALWAYS CLAMP PLATES INTO BOTH SHAKER RACKS AND MAKE SURE TUBES ARE EVENLY DISTRIBUTED ACROSS PLATES.  Shake at 500-700, 1x setting (1550-1700 rpm) for 20 seconds to 2 minutes (check periodically and don’t grind for longer than necessary).

3)	When tissue is ground to a fine powder, centrifuge plate fast and briefly @ 4000 to get powder off lids.

4)	Add 500 uL of Extraction Master Mix to each tube and dip in water bath to thaw. For tubes, shake until buffer is thawed and beads are mobile.  For plates, return to Genogrinder for 10 seconds at 500-700, 1x setting (1550-1700 rpm).

5)	Incubate tubes for 20 minutes in 60 degree C water bath. VERY IMPORTANT: Invert tubes/plates every ~5 minutes during incubation.  Turn off water bath when you are done!

6)	Centrifuge for ~10 seconds at 4000 to separate solids.

7)	Transfer 400 uL liquid from each tube to a new 96-well Costar Plate. Reserve tubes with beads.  [*Clean beads for reuse with soap and water, then rinse with bleach or ethanol and autoclave.]  *this can be done later.

8)	Cool plate to room temperature. Add 400 uL of chloroform to each tube and place in the genogrinder on 0.5x rate at 00 (lowest setting) for 5 minutes.

9)	Centrifuge for 10 minutes at 4000. A band of tissue debris will separate the aqueous (upper) and chloroform (lower) layers.

10)	 Carefully pipet off 280 uL of the aqueous layer and transfer to a new 96-well plate. Avoid drawing up debris or chloroform (if so, recentrifuge briefly and transfer again).

11)	Add 280 uL isopropanol and mix well by inverting 10 times. Place in -20 degree freezer for 20 minutes or as long as overnight.

12)	Centrifuge for 10 minutes at 4000. A grayish gelatinous pellet should form.

13)	Pour off supernatant, using a Kimwipe or paper towel to wick out as much as possible from tube.

14)	Add 200 uL 70% ethanol. Flick tubes multiple times to mix (Do not Vortex).

15)	Centrifuge at 4000 for 10 minutes.

16)	Pour off ethanol, using a Kimwipe or paper towel to wick out as much as possible from tube.

17)	Place open tubes in a 37 degree C incubator overnight to dry. If no incubator is available, tubes can be left out on bench to dry.  Make sure all ethanol is gone before proceeding to next step.

18)	Add 50-100 uL TE or distilled water to each tube. Flick tube to mix well.

CTAB Leaf Buffer (Recipe to make 100 mL Buffer)
 * 1 M Tris Buffer, pH 8.0 		10 mL
 * NaCl					8.3 g
 * EDTA					0.744 g
 * CTAB					2 g
 * Polyvinyl-Pyrrolidone (PVP)		2 g
 * Asorbic acid				0.088 g

Needed for Extraction:
 * Genogrinder
 * Centrifuge (Plate Centrifuge if you are using plates)
 * B-mercaptoethanol
 * Isopropanol
 * 70% Ethanol
 * Chloroform
 * Liquid Nitrogen
 * Fume Hood
 * CTAB Leaf Buffer
 * Water Bath
 * Incubator (optional)