Customer Developed Protocol

CTAB Protocol for Isolation DNA from Plant
TissueContributed by:Allen Van Deynze, Van Deynze Lab, University of California, DavisCUSTOMER DEVELOPED PROTOCOLCTAB Protocol for Isolation DNA from Plant Tissue

Contributor Research Profile
The Van Deynze lab at UC, Davis is a Plant Molecular Biology, Biochemistry and Genomics lab that works onelucidating the basis of capsaicin synthesis and resistance to phytophthora in pepper.
Read more about the Van Deyzne Lab: http://biosci3.ucdavis.edu/Faculty/Profile/View/14262
Contact: avandeyzne@ucdavis.edu

ReferenceThis protocol was used in:Amanda M. Hulse-Kemp, et al., Reference Quality Assembly of the 3.5 Gb genome of Capsicum annuumfrom a Single Linked-Read Library, Biorxiv. 2017 June 20.
https://www.biorxiv.org/content/early/2017/06/20/152777

10x Genomics Products
10x Chromium™ de novo Assembly Solution - https://www.10xgenomics.com/assembly/

Notices
THIS PROTOCOL IS NOT SUPPORTED BY 10X GENOMICSThis Customer Developed Protocol is provided for general information only and is not directly supported,endorsed, or certified by 10x Genomics. 10x Genomics gives no warranties and makes no claims about theprovided protocol.
For questions or to share protocol adjustments, please visit the 10x Community at community.10xgenomics.com
or contact the contributor at avandeyzne@ucdavis.edu.

Customer Developed Protocol

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CUSTOMER DEVELOPED PROTOCOLCTAB Protocol for Isolation DNA from Plant Tissue

Required Buffers and Reagents
1. AMPure XP Beads (Beckman Coulter)
2. DNA Extraction Buffer:
100 mM Tris (pH 8.0)
20 mM EDTA
2% CTAB
1.2 M NaCl
0.1% ß- mercaptoethanol (Add before use. After adding ß- mercaptoethanol, the Extraction Buffer must
be used within a day)

2. Chloroform-Isoamylalcohol (CIA) 24:1 (v/v)
3. Dilution Buffer:
100 mM Tris Buffer (pH 8)
20 mM EDTA
2% CTAB

4. High Salt TE:
10 mM Tris (pH 8)
2 mM EDTA
1 M NaCl (add RNase A at 50 μg/ml)

6. Wash Buffer:
Add 300 ml TE to 700 ml ethanol

7. TE:
10 mM Tris
1 mM EDTA (pH 8)

DNA Extraction Protocol
1. For freeze-dried tissue, grinding is done using a disposable microcentrifuge pestle directly in a 2 mlmicrocentrifuge tube (~200 mg tissue). Keep samples cold until the DNA Extraction Buffer is added. Forseeds, grinding is done using a Geno/Grinder 2010 by placing one 5 mm tungsten ball into a roundedbottom 2 ml microcentrifuge tube.
2. Add 750 μl Extraction Buffer to the ground tissue using a multichannel pipette.
3. Cap the tubes and mix well. Incubate at 60°C for 1 hour. Mix the tubes gently every 10-15 minutes
during the incubation period.
4. Cool the tubes to room temperature for 5 min, then add 750 μl Chloroform:Isoamylalcohol (24:1, v/v) toeach tube, mix well and centrifuge at 3000 g for 15 minutes.
5. Transfer the aqueous layer (~500 μl) to a new set of labeled tubes.

Customer Developed Protocol

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CUSTOMER DEVELOPED PROTOCOLCTAB Protocol for Isolation DNA from Plant Tissue
6. Add 1 ml Dilution Buffer to the aqueous phase.
7. Mix well and incubate at 60°C for ~30 minutes. (Note: Significant amounts of precipitated DNA-CTAB
complex should be observed at the end of incubation).
8. Centrifuge at 4500 g for 15 minutes and discard the supernatant.
9. Add 1 ml Wash Buffer to the pellet and soak it in at room temperature for 30 minutes to remove anyexcess CTAB.
10. Centrifuge at 4500 g for 15 minutes and discard the supernatant.
11. Re-suspend the DNA pellet in 100 μl high salt TE with RNase A and incubate at 60°C for 30 minutes.
12. Transfer the high salt TE DNA solution to a 96-well microtiter plate, one sample per well.
13. Add 1X volume of AMPure XP beads (i.e. 100 μl sample + 100 μl AMPure XP beads).
14. Mix well by pipetting up and down.
15. Incubate the plate for 10-15 minutes.
16. Place on a magnetic stand for 2-5 minutes (or until clear) to collect beads.
17. Gently remove the solution without disturbing the bead pellet.
18. Add 150 μl 75% ethanol solution (keep plate on the magnet).
19. Wait 30 seconds and remove ethanol without disturbing the beads.
20. Repeat the ethanol wash and then let the beads air dry for 2-5 min (make sure you do not over-dry thebeads as it will make re-suspension harder).
21. Add 100 μl Tris/H2O to each sample to re-suspend the DNA, mix well by pipetting up and down.
22. Incubate plate for 10-15 minutes.
23. Place on a magnetic stand for 2-5 minutes (or until clear) to collect beads.
24. Transfer the solution into a new 96-well plate (stock DNA).

THIS PROTOCOL IS NOT SUPPORTED BY 10X GENOMICSThis 10x Community Customer Developed Protocol is provided for general information only andis not directly supported, endorsed, or certified by 10x Genomics. 10x Genomics gives no warranties and makes
no claims about the provided protocol.
For questions or to share protocol adjustments, please visit the 10x Community at community.10xgenomics.com
or contact the contributor at avandeyzne@ucdavis.edu.

Customer Developed Protocol

COMMUNITY.10XGENOMICS.COM