TITLE:  Mimulus sp. CTAB Low Salt Nucleic Acid Prep
AUTHORS: Dylan Moxley, Mackenzie Urquhart-Cronish, Amy Angert

[DESCRIPTION]
This protocol is an adaptation of Xin & Chen (2012) and Arseneau et al. (2017). From the high salt suspension step, users can follow Xin & Chen (2012) protocol to completion for quick clean-up in a 96-well format. If there is no access to or desire for bead-based cleanup, users can follow the protocol outlined by Arseneau et al. (2017) for the DNA pellet and washing steps. 

It is suggested for use with less than ideal samples (mature fresh leaf with high secondary metabolites, field collection silica dried young leaves, oven-dried leaves). Young Mimulus leaf material generally passes in-house QC using a high salt + CTAB method. I recommend starting with Yuan & Sweigart (2019) or Fishman (2020) Mimulus protocols on Protocol.io and then trying this protocol for challenging samples.


This protocol is designed for individual tube format. Users can comfortably complete 96 samples in 3 days using a stacked method (Day 1 Extract, Day 2 Extract/Purify, Day 3 Purify/Quantify). 96 samples are possible in a 2-day format.  The volumes are adapted to make this process efficient, utilizing individual tubes (the use of a combi tip during washes and a larger aqueous phase to draw from during organic phase separation).

Populations with higher trichome density or purple hue (pigmentation) in leaves were observed to be more challenging to extract cleanly using a standard CTAB protocol in my hands. This protocol was able to handle these samples, along with 2-year-old oven-dried mature leaf tissue. 

Modifications:
CTAB-DNA complex incubation time is increased to 2 hours, including a cool-down step meant to flocculate the complexes. This can be shortened to 1 hour, depending on your sample. Once the solution begins to precipitate CTAB-DNA complexes, it is fine to begin the spin. High-yielding samples are usually ready in 1 hour of incubation. If the sample fails to precipitate after 2 hours, spin anyway, as a small pellet will likely form. In this case, reduce the final elution volume to 35 uL. 

CTAB-DNA centrifugation is increased (3k rcf → 6k rcf) to stabilize the pellet during decanting (pour-off).

DNA-CTAB precipitation centrifugation has increased (3k rcf → 10k rcf) before the high salt suspension for higher yields without sacrificing purity. 

Two rounds of desalting are used before the final elution. Using one wash yields 260/230 ~ 1.6-2.0, while two washes are generally >2.0. 

Average nanodrop 260/280 1.9 + 0.16 , 260/230 2.16 + 0.3 across all 3 tissue types used n = 690.

[STEPS]
SECTION: Tissue Prep
3. Flash freeze in liquid nitrogen and load into tube mix mill grind plates chilled to -20 °C
 


SECTION: Tissue Prep
4. Grind 45 s 30/sec and gently knock tissue until free in the tube. Check how well the tissue is ground (should still be frozen).
 

SECTION: Tissue Prep
4.1. If needed, flash freeze again and flip the orientation of grind plate (tubes closest to the machine to the further position away from the matching), repeat grind until well milled

SECTION: Tissue Prep
5. When ready to extract, spin on a short cycle [ 2-3 sec to ~ 2500 rcf (g) ] to collect tissue from the cap. 
SECTION: Tissue Prep
4.2. Store at -20 °C or proceed directly to extraction. I prefer to load & grind the samples the day before extraction.

SECTION: Tissue Extraction
6. For 48 samples, prepare 40 mL CTAB Extraction Buffer + 40 µL Beta-mercaptoethanol (0.1%) + 100 µL Proteinase K (0.1mg/mL) under the fume hood and warm to 60 °C in a waterbath. 

                
  
 



SECTION: Tissue Extraction
7. Under the fume hood, dispense 750 µL warmed CTAB buffer solution (w/ B-mercap) per sample using a p1000 pipette, changing the tip every 6-8 samples or if  bubble forms in the tip
 
. 

SECTION: Organic Phase Seperation
11. Immediately load and spin samples 3000 rcf, 15 min 

SECTION: Tissue Extraction
8. Vortex samples under the fume hood for 30-60 seconds or until the tissue is dispersed in the buffer.

SECTION: Tissue Extraction
9. Incubate in a water bath 60 °C , inverting several times gently every 20 minutes to ensure good contact between buffer and tissue. Let tubes cool in the fume hood for 5 minutes

SECTION: Organic Phase Seperation
10. Add equal volume 750 µL Chloroform: Isoamyl alcohol 24:1 to 24 samples. The other 24 samples can remain at room temperature in the fume hood until ready. 



SECTION: Tissue Prep
2. Load tissue loosely into a 2.0mL tube with 2x 3.2 mm chrome steel beads (Biospec, cat. 11079132c)  

50 mg 
or
100 mg 

SECTION: Organic Phase Seperation
10.1. Close tubes, place a paper towel, and then a tube block on top. Shake samples by hand (5-7/sec) between the two blocks for 20 s under the fume hood.
 


SECTION: Organic Phase Seperation
10.2. Alternatively, return samples to mix mill  20 s at 7/s

SECTION: Organic Phase Seperation
12. Transfer 500 µL into the 1.5 mL tube containing CTAB Dilution Buffer using a p1000 pipette. Mix 1 -2 times by pipette. If interphase is disturbed, recentrifuge sample 3000 rcf, 3 min.
 
 
 

 
 


SECTION: Organic Phase Seperation
11.1. In the first 10 minutes, dispense 1.5 mL tubes with 1000 µL CTAB Dilution Buffer
 


SECTION: Set-up
1. For each sample label:
 1x 2.0 mL tube with 2x 3.2 mm chrome steel beads (Biospec, cat. 11079132c)    
 1x 1.5 mL tube

SECTION: Organic Phase Seperation
13. With the next 24 samples   and repeat. 
 


SECTION: DNA-CTAB Precipitation
14. Incubate samples in a water bath or heat block 65 °C 

SECTION: DNA-CTAB Precipitation
15. Place samples on shaker   or until the solution looks saturated in small 'crystals'.
 

 

SECTION: DNA-CTAB Precipitation
16. Spin samples 6000 rcf, 15 min 

SECTION: DNA-CTAB Precipitation
17. Pour off supernatant gently and completely. If pellet detaches, stop pouring and respin for 5 min before finishing pour. 

 

SECTION: DNA-CTAB Purification
18. Add 500 µL Wash Buffer and invert several times until pellet detaches from the wall and begins to dissolve. 

 

SECTION: DNA-CTAB Purification
19. Put on shaker   

SECTION: DNA-CTAB Purification
20. Spin tubes 12000 rcf, 15 min 


 

SECTION: DNA High Salt RNASE Treatment
23. Gently suspend pellet in 100 µL High Salt TE (1M) + 50 ug/mL final concentration RNASE (0.25 uL/100 uL)

  

 

SECTION: DNA High Salt RNASE Treatment
24. Incubate either 37 °C  and store 4 °C OR -20 Room temperature 

 

SECTION: DNA-CTAB Purification
21. Pour off supernatant gently and completely. Dab tubes on an autoclaved paper towel to remove trace liquid
 
 

SECTION: DNA-CTAB Purification
22. Dry tubes open 37 °C 15-20 min or until tubes are just dry.
 


SECTION: DNA Precipitation
25. Add 500 uL 90% ETOH (final concentration of 75%) 
 

SECTION: DNA Precipitation
26. Invert several times and incubate -20 Room temperature 

SECTION: DNA Precipitation
27. Spin 16000 rcf, 20 min 

SECTION: DNA Precipitation
28. Pour off supernatant slowly and completely. 
SECTION: DNA Purification
29. Desalt the pellet using 500 uL Wash Buffer, flick tube to suspend pellet briefly
SECTION: DNA Purification
30. Spin 16000 rcf, 5 min 

SECTION: DNA Purification
31. Pour off supernatant slowly and completely.
SECTION: DNA Purification
32. If DNA 260/230 of 1.6-1.8 with slightly higher yield is acceptable continue to step 33, else     and repeat once for DNA 260/230 > 2.0 (Genome BC/Quebec QC standard). 

SECTION: DNA Purification
33. Dry tubes open at 37 °C 

SECTION: DNA Elution and Quantification
34. Add 55 uL low TE ( Genome Quebec) or 10mM Tris-Hcl pH 8.0
 

SECTION: DNA Elution and Quantification
35. Incubate   

SECTION: DNA Elution and Quantification
36. Mix samples by pipette (gently!) and transfer 54 µL to a PCR plate

SECTION: DNA Elution and Quantification
37. Access purity of the sample using 2 µL on a  Nanodrop1000/2000

SECTION: DNA Elution and Quantification
38. Quantify 2 µL sample using broad spectrum Qubit kit 
 


SECTION: DNA Elution and Quantification
39. Optional: run 3-5 uL sample on 1.0% agarose gel in TAE buffer, 100V 30 min to check for average molecular weight, and sample integrity.
 
 

SECTION: DNA Precipitation and Elution with paramagentic beads
25. Transfer the 100 µL high salt TE DNA solution to 96-well PCR plate, one sample per well.

 

SECTION: DNA Precipitation and Elution with paramagentic beads
26. Add 5 µL MagAttract suspension G solution to each well using a multichannel pipette.
 


SECTION: DNA Precipitation and Elution with paramagentic beads
27. Add 120 µL 100% ethanol to each well using multichannel pipette.

SECTION: DNA Precipitation and Elution with paramagentic beads
28. Cover the 96-well PCR plate tightly with a silicone plate sealing mat and mix gently in your hands (to allow the DNA to adhere to the beads).
 

SECTION: DNA Precipitation and Elution with paramagentic beads
29. Place the DNA plate on Magnet B to hold the MagAttract beads and then pour off the ethanol solution.
 

SECTION: DNA Precipitation and Elution with paramagentic beads
30. Wash the beads three times with 200 µL washing buffer and, after the final wash, air-dry the beads for 10 min at RT.


 




SECTION: DNA Precipitation and Elution with paramagentic beads
31. Add 55 µL TE to each sample well to re-suspend DNA.
 


SECTION: DNA Precipitation and Elution with paramagentic beads
32. Incubate at 60 °C  5 min and mix gently (to allow the bound DNA release into TE solution).
 



SECTION: DNA Precipitation and Elution with paramagentic beads
33. Place the plate on a magnetic plate and wait 2 minminutes before transferring 54 µL of the elution buffer to a new 96-well plate.

SECTION: DNA Quantification
34. Check the purity of the sample using 2 µL on a Nanodrop1000/2000

SECTION: DNA Quantification
35. Quantify 2 µL sample using broad spectrum Qubit kit 



SECTION: DNA Quantification
36. Optional: run a 3-5 μL sample on a 1.0% agarose gel in TAE buffer at 100V for 30 min to check for molecular weight distribution and sample integrity.