TITLE: DNA extraction | CTAB-chloroform | 96 wells plate 
AUTHORS: Lila Fishman, Simon Joly, Andrea Corkal, Jérôme Burkiewicz

[DESCRIPTION]
This is a 96-well version of the classic CTAB-chloroform plant DNA extraction (Doyle & Doyle 1987), developed by John Willis and Lila Fishman in ~2000 and since optimized by multiple lab groups working on Mimulus (monkeyflowers). It has been used successfully for downstream applications in other plant taxa, including Asclepias, Larix, Cynoglossum, and Populus. In the Joly lab, we used it on Impatiens so far.

Link to the original protocol: dx.doi.org/10.17504/protocols.io.bgv6jw9e

The protocol requires more specialized equipment (see below) than the single-tube version, but allows for inexpensive high-throughput DNA extraction. The yields are sufficient in quantity and quality for next-generation sequencing applications (e.g., ddrads, whole-genome Illumina sequencing), as well as PCR marker genotyping. 

Equipment: 
Genogrinder or similar bead-beater
Benchtop centrifuge w/ deep-bucket plate rotor (e.g., Qiagen Sigma 4-15K)
8-channel pipettor + tips 
Hood w/ waterbath
96-well bead dispenser

Materials and supplies:
dry ice (optional)
liquid N2
3mm grinding balls or "beads"
chloroform
CTAB DNA Extraction Buffer
>95% isopropanol
 70% ethanol
96-well “plates” of Costar 8-tube strips  (Corning#4412, #4418) and strip-caps (Corning#4408)
 

 


[STEPS]
SECTION: Tissue collection
5. Pre-label the tubes
 

SECTION: Tissue collection
6. Pre-load cleaned and autoclaved beads into plate of tubes (could use a bead dispenser if available).

SECTION: Tissue collection
7. Add 10mg of leaves (max. 30 mg) to each tube. Try to be consistent to have a similar yield for each extraction.
 


 

SECTION: Tissue grinding
8. Put the samples in the tissue lyser for 2 min at a frequency of 30 (1/s).

 


SECTION: DNA extraction
10. 
 

 

Turn on water bath in fume hood (60°C) and clear workspace in hood. 
Make sure you have sufficient CTAB buffer and reagents/supplies for all steps before starting.
SECTION: DNA extraction
11. Glove up. 
In hood, measure out 600 µl of CTAB extraction buffer per sample (+8-10%) into a wide trough (or clean tipbox lid)
Add 1 µl b-mercaptoethanol per 600 µl of CTAB extraction buffer, and stir to mix.
Add 4µl of RNAseA per sample.
 

SECTION: DNA extraction
12. Uncap the tubes with the ground samples VERY CAREFULLY/SLOWLY and discard cap strips immediately into waste bin (they are a contamination risk).
Add 600 µl of CTAB buffer (including β-mercaptoethanol and RNAseA) to each sample using multichannel pipettor. 
Re-cap tightly with FRESH caps. 
SECTION: DNA extraction
13. Use the vortex or the tissue lyser to homogenize the sample in the buffer. Make sure there is no powder left at the bottom of the tube. 


SECTION: DNA extraction
14. Incubate tubes for ~20 min. in 60°C water bath. 
Meanwhile, label next set(s) of plates/tube-strips to match your collection plates.
 

SECTION: DNA extraction
15. Post-incubation, remove plates from the water bath and blot them off. Re-tighten caps.
Centrifuge plates for 2 min to pellet solids @4000 rpm. 
SECTION: DNA extraction
16. Transfer all the supernatant possible to a new labelled 96-well Costar plate using multi-channel pipettor. 
 


 

SECTION: DNA extraction
17. Decant chloroform into large dedicated trough (tipbox lid or other HARD plastic).
Add 500 µl of chloroform (equal volume) to each sample tube. 
Re-cap samples (tightly!) with fresh caps. 


 

 

SECTION: DNA extraction
18. Hand-invert for 5 minutes using a paper towel over the lids to catch leaking from the caps.


SECTION: DNA extraction
19. Centrifuge for 15 min @ 4000 rpm to separate aqueous (upper) and chloroform (lower) layers. 

SECTION: DNA extraction
20. While you are waiting on the spin:
Fully label a fresh plate of strip tubes -- these will be final home of samples. 
Arrange the handy-dandy-cut-down-rack for interface-viewing/pipetting (center front) and your freshly labeled Costar plate of tubes (center-back). Put an empty Costar base for waste tubes (to the left, if right-handed).
Set pipettor to 300 mL and SLOW speed (if automated), and get a fresh box of tips for each plate.
SECTION: DNA extraction
21. Bring plate of centrifuged samples to hood. Place on right (if right-handed).
One strip at a time, put a sample strip and the corresponding empty strip in the handy-dandy-cut-down-rack. 
Un-cap sample (can do before, during, or after moving, depending on preference).

Carefully pipet off the aqueous layer (up to 300mL) of sample strip and transfer it the matched empty strip.
Move chloroform waste to left waste plate and return filled strip to new base.
Repeat until all samples are transfered.


 

 

 



SECTION: DNA extraction
22. [Out of hood if you like.]
Add 1.5 volumes of cold 100% isopropanol to each aqueous sample, cap well with fresh strip-caps, and mix by hand- inverting plate once or twice.
(e.g. for a sample of 200uL add 300uL of isopropanol)

 Place in -20 freezer for at least 30 minutes or as long as overnight (we generally do the latter). 

SECTION: DNA extraction
23. Centrifuge for 15 min. at 4000 rpm. 
A greyish gelatinous pellet is often visible in the bottom of the tube (but may not be).
SECTION: DNA extraction
24. Un-cap. To re-use caps (optional):  lay them in order on a trifold towel labelled with plate #, NOT touching.
One strip at a time, gently tilt to pour off isopropanol supernatant (into a dedicated waste tipbox lid).
While each strip is still tilted but mostly empty, blot the open ends with a fresh bit of trifold towel to wick out as much liquid as possible.


SECTION: DNA extraction
25. Add 200 µl cold 70% ethanol (mol. grade) to each sample.
Flick to rinse pellet. It should float up off the bottom of the tube. 
(OR can invert to really get rid of isopropanol on walls. Cap if so)

Centrifuge at 4500rpm for 5 min (no need to re-cap yet, but you can)

Pour off ethanol as with isopropanol, wicking out as much as possible with paper towel at the end of the pour.
 

SECTION: DNA extraction
26. Air-dry the pellet for 1-2 hours at room temp. with a Kimwipe placed over the open tubes. If you have to leave for longer, re-cap loosely and dry later. Don't overdry.


SECTION: DNA extraction
27. Once pellets are dry (no ethanol smell), add 50μL TE buffer to each sample.
Cap with retained or new caps. 
Flick tube-strips to mix well and/or leave out at room temperature for at least an hour to fully re-suspend.
Refrigerate if diluting/using wihtin a few weeks, otherwise store in -20 or -80. (Do not freeze before pellet has fully resuspended!)
 


SECTION: Sample drying
2. Turn on the freeze dryer and the blue pump attached to the side. The freeze dryer will take 30 minutes to reach the proper conditions.
SECTION: Sample drying
3. When the machine is ready, it should say "Load/Unload" in the top left corner, load the samples that have been stored at -80C. Click "Start" and the machine. Let the machine run for 48 hours.

 

SECTION: Sample drying
1. Put the samples to be freeze-dried at -80 and let them sit for at least one hour.
SECTION: Sample drying
4. After 48 hours, click "Aerate" to stop the machine and remove the red cap from the tube on the left side to drain the moisture collected. Unload the samples. 
SECTION: Tissue grinding
9. Centrifuge the samples for 2 min @2000rpm to get powder off caps and down into bottom of the tubes.