Jun 16, 2025

Public workspace Mimulus sp. CTAB Low Salt Nucleic Acid Prep

DOI
https://dx.doi.org/10.17504/protocols.io.261ge43zyv47/v1
  • Dylan Moxley1,
  • Mackenzie Urquhart-Cronish2,
  • Amy Angert2
  • 1Department of Botany and Biodiversity Research Centre, University of British Columbia, Vancouver, BC, Canada;
  • 2Department of Zoology and Biodiversity Research Centre, University of British Columbia, Vancouver, BC, Canada
Dylan Moxley
Icon indicating open access to content
QR code linking to this content
DOI: https://dx.doi.org/10.17504/protocols.io.261ge43zyv47/v1
Protocol Citation: Dylan Moxley, Mackenzie Urquhart-Cronish, Amy Angert 2025. Mimulus sp. CTAB Low Salt Nucleic Acid Prep. protocols.io https://dx.doi.org/10.17504/protocols.io.261ge43zyv47/v1
Manuscript citation:

License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
This protocol was used for Urquhart-Cronish et al. (2025) and Anstett et al. (2024-bioRxiv)
Created: November 25, 2020
Last Modified: June 16, 2025
Protocol Integer ID: 44878
Keywords: Mimulus, DNA, Dry Leaf, Fresh Leaf, CTAB, Difficult Tissue, young mimulus leaf material, dna pellet, high salt suspension for higher yield, mimulus protocol, high salt suspension step, high salt suspension, protocol for challenging sample, standard ctab protocol, dna complex incubation time, ctab method, dried mature leaf tissue, ctab precipitation centrifugation, utilizing individual tube, individual tube format, fresh leaf with high secondary metabolite, individual tube, high salt, dna centrifugation, challenging sample, dried leaf, mature leaf tissue, sacrificing purity
Abstract
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.
Materials
Equipment

Fume hood

-20C freezer

Liquid nitrogen dewar
Liquid nitrogen 2L plastic dewar flask (Thermofisher Nalgene 4150-2000)
Metal Sample basket (for dewar flask)
Mix mill/bead beater
Waterbath or heat block
Eppendorf 5424 or any centrifuge capable of 16,000 rcf with MCT rotor
Shaker (with temp control is ideal)

p1000 pipette
p200 pipette
p2 pipette
Eppendorf Repeater M4 (optional)
Expendables
p1000 tips
p200 tips
p2 tips
25 mL Orange Combitip

2.0 mL MCT tubes (Fisherbrand 05-048-138)
1.5 mL MCT tubes (Fisherbrand 05-408-129)
3.2 mm chrome steel beads (Biospec, cat. 11079132c)

Qubit approved assay tube 0.5 mL (Invitrogen Q32856)

Eppendorf twin.tec, Full Skirt (Cat# 951020401)
VWR Aluminum Foil for PCR (Cat# 60941-074)
Chemicals & Reagents

Liquid Nitrogen 1-2 L

Enzymes
ReagentRNase A (10 mg/mL)Thermo Fisher ScientificCatalog #EN0531

CTAB
ReagentHexadecyltrimethylammonium bromide (CTAB)Merck MilliporeSigma (Sigma-Aldrich)Catalog #H9151
Reagent1M Tris-HCl (pH 8.0)Thermo Fisher ScientificCatalog #15568025
ReagentUltraPure 0.5M EDTA, pH 8.0Thermo Fisher ScientificCatalog #15575-038
ReagentNaClMerck MilliporeSigma (Sigma-Aldrich)Catalog #53014
Reagentb-mercaptoethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M3148-25ML
ReagentAmbion Proteinase KThermofisherCatalog #AM2546
Organic Extraction
ReagentChloroformMerck MilliporeSigma (Sigma-Aldrich)Catalog #366919-1L
Reagentisoamyl alcoholMerck MilliporeSigma (Sigma-Aldrich)Catalog #W205702
Wash Buffer / Precipitation
ReagentEthanol

Quantification
ReagentQubit™ dsDNA BR Assay KitThermo FisherCatalog #Q32850

Use at minimum distilled water for stock prep, ultrapure recommended for TE elution buffer
ReagentUltraPure Distilled Water Invitrogen - Thermo FisherCatalog #10977-015

Stocks

5M NaCl
AB
75 mLDistilled Water
58.44 gNaCl
200 mLFinal Volume
Add salt slowly (3-5g) while mixing thoroughly between additions. Add water as the solution becomes saturated. Complete volume in a volumetric flask. Autoclave 20 min hold liquid cycle.

Te Buffer (Genome Quebec)
ABC
490 mLDistilled Water (Ultrapure/Milli-Q preferred)
5 mL10 mM1M TRIS pH 8.0
100 uL0.1 mM0.5M EDTA pH 8.0
500 mLFinal Volume
Add in order, finalize volume in a volumetric flask

Te High Salt (Xin & Chen, 2012)
ABC
50 mLDistilled Water (Ultrapure/Milli-Q preferred)
1 mL10 mM1M TRIS pH 8.0
200 uL1.0 mM0.5M EDTA pH 8.0
20 mL1.0 M5M NaCl
100 mLFinal Volume
Add in order, mixing between additions. Finalize volume in a volumetric flask.

Wash Buffer (Xin & Chen, 2012)
AB
70 mLEthanol, molecular biology grade
vol toTE Buffer
100 mLFinal Volume
Mix day of extraction, discard after 2 week

CTAB Extraction & Dilution Buffer (Xin & Chen, 2012)
ABC
50 mLDistilled Water (start with 75 mL for Dilution Buffer)
10 mL100 mM1M TRIS pH 8.0
4 mL20 mM0.5M EDTA pH 8.0
24 mL1.2 M5M NaCl (omit for Dilution Buffer)
2 g2% w/vCTAB
100 mLFinal Volume
Add in order.
Once CTAB is added, mix gently on a stir plate for 5 min 200-300 rpm (too high will cause foam) until any chunks break apart
Heat in a 60-65C water bath for 5 min, lightly swirling to fully dissolve CTAB.
Complete volume in a volumetric flask (pour slowly down the sidewall!!).
Return to stir plate for 5 minutes to ensure well mixed.
Optional: Autoclave liquid cycle 20 min - I do not because all liquid stocks are already sterilized/autoclaved and CTAB grade is purified for molecular biology

Ideally use within 14 days (stable for months though)




Protocol materials
ReagentNaClMerck MilliporeSigma (Sigma-Aldrich)Catalog #53014
Reagentb-mercaptoethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M3148-25ML
ReagentChloroformMerck MilliporeSigma (Sigma-Aldrich)Catalog #366919-1L
ReagentRNase A (10 mg/mL)Thermo Fisher ScientificCatalog #EN0531
ReagentUltraPure 0.5M EDTA, pH 8.0Thermo Fisher ScientificCatalog #15575-038
ReagentHexadecyltrimethylammonium bromide (CTAB)Merck MilliporeSigma (Sigma-Aldrich)Catalog #H9151
ReagentQubit™ dsDNA BR Assay KitThermo FisherCatalog #Q32850
Reagent1M Tris-HCl (pH 8.0)Thermo Fisher ScientificCatalog #15568025
ReagentAmbion Proteinase KThermofisherCatalog #AM2546
Reagentisoamyl alcoholMerck MilliporeSigma (Sigma-Aldrich)Catalog #W205702
ReagentUltraPure Distilled Water Invitrogen - Thermo FisherCatalog #10977-015
ReagentEthanol
Troubleshooting
Set-up
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

Tissue Prep
1h
Load tissue loosely into a 2.0mL tube with 2x 3.2 mm chrome steel beads (Biospec, cat. 11079132c)

Amount50 mg dried tissue
or
Amount100 mg fresh leaf

Flash freeze in liquid nitrogen and load into tube mix mill grind plates chilled to Temperature-20 °C
Equipment
Mix Mill (MM 300)
NAME
Mix Mill
TYPE
Retsch
BRAND
23392
SKU
https://americanlaboratorytrading.com/lab-equipment-products/qiagen-retsch-mm300-tissuelyser-lab-vibration-mill-mixer_11444
LINK


Grind Duration00:00:45 30/sec and gently knock tissue until free in the tube. Check how well the tissue is ground (should still be frozen).
Ground & collected Mimulus leaf

45s
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

Store at Temperature-20 °C or proceed directly to extraction. I prefer to load & grind the samples the day before extraction.

When ready to extract, spin on a short cycle [ 2-3 sec to ~ 2500 rcf (g) ] to collect tissue from the cap.
Tissue Extraction
1h 30m
For 48 samples, prepare Amount40 mL CTAB Extraction Buffer + Amount40 µL Beta-mercaptoethanol (0.1%) + Amount100 µL Proteinase K (0.1mg/mL) under the fume hood and warm to Temperature60 °C in a waterbath.

Reagentb-mercaptoethanolMerck MilliporeSigma (Sigma-Aldrich)Catalog #M3148-25ML
ReagentAmbion Proteinase KThermofisherCatalog #AM2546
Note




Under the fume hood, dispense Amount750 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
Note
Optional: Combitip 25mL step level 1.5 Eppendorf Repeater M4

When dispensing CTAB there are few things to note.
1. I like to 'prime' the pipette by drawing the solution up and down once using the first stop of the pipette, then drawing up the volume a second time. This is only the first time you use a fresh tip.

2. Do not go to the second stop on the pipette (air expel) if you are planning to reuse the tip. Doing so will create bubbles that can be sucked into the pipette body. For inexperienced users, consider starting with filter tips to protect the pipette until comfortable.

3. Warmed solutions can often be overdrawn (think of the effect of temperature on meniscus shape and height-of-rise). Pay attention to make sure the volume is not 'creeping' up the pipette if reusing the tip (generally I found every 6-8 samples needed a new tip for accuracy).


.

Vortex samples under the fume hood for 30-60 seconds or until the tissue is dispersed in the buffer.

Incubate in a water bath Temperature60 °C 60 min , 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

Organic Phase Seperation
45m
Add equal volume Amount750 µL Chloroform: Isoamyl alcohol 24:1 to 24 samples. The other 24 samples can remain at room temperature in the fume hood until ready.



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 Duration00:00:20 under the fume hood.
Note
Preferred for speed of loading to centrifuge and keeping chloroform under the hood.


20s
Alternatively, return samples to mix mill Duration00:00:20 at 7/s

20s
Immediately load and spin samples Centrifigation3000 rcf, 00:15:00

In the first 10 minutes, dispense 1.5 mL tubes with Amount1000 µL CTAB Dilution Buffer
Note
NaCl molarity of CTAB extraction buffer is 1.2M, diluting reduces molarity to < 0.5M and precipitates DNA-CTAB complexes leaving polysaccharides in solution (Murray & Thompson 1980; Xin & Chen 2012)


Transfer Amount500 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 Centrifigation3000 rcf, 00:03:00 .
2 mL tube with three phases separated out after centrifuging and before transferring 500 ul of the supernatant (top aqueous layer) to the 1.5 mL tube containing CTAB dilution buffer. This sample is from desiccant dried young leaf tissue.
Note
Place the tip just below the surface of the top aqueous phase, slowly moving down the tube while drawing up the solution. There is enough aqueous phase (~650 - 750ul ) that no interphase should be disturbed when using a single draw. If air bubbles occur, dispense the volume into the 1.5 ml tube, and then draw up another small portion to complete the volume to the 1.5 ml mark.

Top phase added to CTAB Dilution Buffer. Left: oven-dried young leaf, center: fresh mature leaf, right: fresh young leaf. All three resulted in clean DNA with sufficient yield.


With the next 24 samples Go to and repeat.
Note
OPTIONAL: I like to begin the next 24 samples at the 2 minutes left mark on the spin cycle. The centrifuge is done by the time I finish dispensing chloroform. I unload the 1st batch, then shake and load the 2nd batch. During the 2nd batch spin, I transfer the top phase of the 1st batch.


DNA-CTAB Precipitation
2h 30m
Incubate samples in a water bath or heat block Temperature65 °C 60 min

Place samples on shaker Shaker150 rpm, Room temperature , 01:00:00 or until the solution looks saturated in small 'crystals'.
Note
For most extractions, the solution will look saturated. If DNA concentrations are high enough, these 'crystals' will flocculate into long strands and drop out leaving the solution clear with DNA-CTAB 'clumps' at the bottom. These yields tend to be >2.5-3 ug. This solution has yielded up to 7.5 ug.

CTAB Precipitation Post Shaking. Left: "crystal" saturated sample (average appearance is like a hazy IPA) Right: formation of flocculate "strands" forming, note larger 'crystal' size above the aggregate.

Spin samples Centrifigation6000 rcf, Room temperature, 00:15:00

Pour off supernatant gently and completely. If pellet detaches, stop pouring and respin for 5 min before finishing pour.

CTAB-DNA pellets. Left: oven-dried young leaf, Right: fresh mature leaf

DNA-CTAB Purification
1h 30m
Add Amount500 µL Wash Buffer and invert several times until pellet detaches from the wall and begins to dissolve.

Note
For faster dispensing, a 25 mL Combitip with step level 1 (500 uL) will fill 48x samples in under 3 minutes

This step cleans up contamination from the DNA-CTAB complexes

Put on shaker Shaker150 rpm, Room temperature , 00:30:00

Spin tubes Centrifigation12000 rcf, Room temperature, 00:15:00


Washed CTAB-DNA pellet. Left: dried leaf, Center: fresh mature leaf, Right : fresh young leaf

Pour off supernatant gently and completely. Dab tubes on an autoclaved paper towel to remove trace liquid
Note
If time-constrained, pipette off remaining traces of wash buffer using p200 pipette

Dry tubes open Temperature37 °C 15-20 min or until tubes are just dry.
Note
Do not over-dry tubes, it will become difficult to resuspend the pellet and can shear DNA.


DNA High Salt RNASE Treatment
Gently suspend pellet in Amount100 µL High Salt TE (1M) + 50 ug/mL final concentration RNASE (0.25 uL/100 uL)


Note
Pellet can be suspended gently by pipette, or on shaker RT, 150 RPM, 30 minutes.

This step disassociates CTAB from DNA by introducing NaCl > 0.5M, along with degrading RNA.

For 48 samples, prepare 5mL TE Buffer 1M NaCl + 12.5 uL Purelink RNASE

Incubate either Temperature37 °C 60 min and store Temperature4 °C overnight OR TemperatureRoom temperature overnight

Note
This is usually my stopping point for the day. Another user reported good success with 37C for 1 hour and longer-term storage at -20C.

From this point, can either follow either alcohol precipitation and desalting (Arseneau), or use of paramagnetic beads (Xin and Chen). Personal preference is to follow Xin and Chen if Suspension G beads are available.

Step case

Arseneau (2017)
15 steps

DNA Precipitation
1h 45m
Add 500 uL 90% ETOH (final concentration of 75%)
Note
For faster dispensing, a 25 mL Combitip with step level 1 (500 uL) will fill 48x samples in under 3 minutes


Invert several times and incubate TemperatureRoom temperature 60 min

Spin Centrifigation16000 rcf, Room temperature, 00:20:00

Pour off supernatant slowly and completely.
DNA Purification
45m
Desalt the pellet using 500 uL Wash Buffer, flick tube to suspend pellet briefly
Spin Centrifigation16000 rcf, Room temperature, 00:05:00

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

Dry tubes open at Temperature37 °C 15-20 min

DNA Elution and Quantification
Add 55 uL low TE ( Genome Quebec) or 10mM Tris-Hcl pH 8.0
Note
Genome Quebec TE: 10 mM Tris-HCl pH 8.0, 0.1 mM EDTA
Standard TE (Cold Harbour Springs): 10 mM Tris-HCL pH 8.0, 1 mM EDTA

Incubate Shaker150 rpm, 37°C, 00:30:00

Mix samples by pipette (gently!) and transfer Amount54 µL to a PCR plate

Access purity of the sample using Amount2 µL on a Nanodrop1000/2000

Quantify Amount2 µL sample using broad spectrum Qubit kit
Note
ug/mL can be converted to ng/uL by multiplying *10 for 2 uL volume, (i.e 0.280 ug/ml = 28.0 ng/uL and 1.19 ug/mL = 119 ng/uL) This saves clicking calculate stock concentration everytime between reading samples.




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.
1% agarose TAE buffer electrophoresis gel. 100V 30 min. Left 2 lanes: fresh mature leaf, Center: Generuler 1kb plus, Right: 2 year old oven dried leaf

Protocol references
Xin & Chen (2012)-
10.1186/1746-4811-8-26
Arseneau et al. (2017) -  
https://doi.org/10.1111/1755-0998.12616