Supplemental Protocol:
CKC protocol for isolation of total nucleic acid
Informed by: Li et al, 2008; Echevarria-Machado et al, 2003.
Brief protocol:
Prep: 10ml f2+k; store in dark, 1 month at RT
buffer f2
7ml
5M KOAc
3ml
β-Merc.Et
100µl
1.
Add 100mg tissue to 2ml screw-cap tube on ice. Add 1ml f2+k, two ¼” beads, and tightly
cap. Chill -20° 7min. Option: to reduce chances of tube failure, thaw on ice for 1-2 min. before
proceeding to FastPrep.
2.
FastPrep: speed=4.0 for 30-60s. Inspect tubes for cracks.
3.
65° 8min; immediately proceed to step 4.
4.
Spin 10K rcf, 5 min.
5.
Transfer 625µl of supernatant to a 1.5ml tube and add equal volume chloroform/isoamyl
alcohol (24:1, v/v); vigorously shake 15-20s, or vortex.
6.
Spin 10min 15k rcf; add aqueous phase (430µl) to new 1.5ml tube.
7.
Add 17.5µl (~0.04 volumes) 3M KOAc pH 5.2; pipette mix.
8.
Add 0.7 volumes (313µl) isopropanol; mix by pipette.
9.
Transfer to ES RNA column and spin 30s, ≥ 10K rcf. Discard flow-through.
Note: If working with volumes greater than those suggested, transfer a portion of the
solution to the column, spin, discard the solution and repeat with remaining solution.
10. Add 500µl Wash Buffer, spin 30s, ≥ 10K rcf; discard flow-through.
11. Add 500µl Wash Buffer, spin 1min, ≥ 10K rcf; discard flow-through.
12. Spin 2min ≥ 10K rcf w/empty (optional: new) collection tube.
13. Transfer column to 1.5ml tube; add 65µl H2O (RNase-free) to membrane; rest 2-5min. spin
1min ≥ 10K rcf.
1
Supplemental Protocol:
CKC protocol for isolation of total nucleic acid
Informed by: Li et al, 2008; Echevarria-Machado et al, 2003.
Note: take usual precautions for preventing RNase contamination of samples. This protocol is
designed to isolate total nucleic acid (DNA and RNA together). It can be modified to isolate
either RNA or DNA only but such modifications have not been thoroughly tested.
Prep: 10ml f2+k; store in dark, 1 month at RT
buffer f2
7ml
5MKOAc
3ml
β-Merc.Et
100µl
Note: β-Merc.Et may be added fresh, if desired; can be stored in solution ~1 month.
1.
Add 100mg tissue to 2ml screw-cap tube on ice. Add 1ml f2+k, two ¼” beads, and tightly
cap. Chill -20° 7min. Option: to reduce chances of tube failure, thaw on ice for 1-2 min.
before proceeding to FastPrep; if using Tough Tubes, this brief thaw is not necessary.
Note: do not exceed 100mg of tissue; more tissue does not equal higher yield.
Note: f2+k should be left at room temp until it is added to tubes. CTAB will precipitate
as solution cools. f2+k may also become too viscous to accurately pipette if stored on ice.
2.
FastPrep: speed=4.0 for 30-60s. Inspect tubes for cracks.
Note: if tubes are cracked, decant/pipette to new tube.
Note: 60s of FastPrep time will more thoroughly disrupt tissue; a speed setting of 4.5 may
help with disruption of particularly tough tissue. Longer times and higher speeds may
compromise the sample and fracture less durable tubes.
Note: if FastPrep is not available, alternate methods of disruption/homogenization may
be used. FastPrep is faster than mortar/pestle liquid N and, in limited testing, produces
higher yields.
Note: because f2+k does not completely inactivate RNase, it is important to proceed
through step 5 as reasonably fast as possible.
3.
65° 8min; immediately proceed to step 4.
4.
Spin 10K rcf, 5 min. (rcf = relative centrifugal force).
5.
Transfer 625µl of supernatant to a 1.5ml tube and add equal volume chloroform/isoamyl
alcohol (24:1, v/v); vigorously shake 15-20s, or vortex.
Note: some plants will produce flocculate debris that does not pellet well; wide bore tips
may facilitate transfer.
Note: be sure tubes are tightly capped before shaking.
2
Note: More solution can be transferred (with respective increases in following steps).
6.
Spin 10min 15k rcf; add 430µl of aqueous phase to new 1.5ml tube.
Note: If transferring more than 430µl, adjust volumes in subsequent steps accordingly.
7.
Add 17.5µl (~0.04 volumes ) 3M KOAc pH 5.2; pipette mix.
Note: at this point, pH should drop to ≤ 7.4. Nucleic acids bind to columns less efficiently
at pH > 7.5. Optional: to confirm shift in pH, first add 1µl 50mM phenol red. Phenol red
will shift from red to yellow as acidic KOAc is added.
8.
Add 313µl (0.7 volumes) isopropanol; mix by pipette.
Note: precipitate may form and should dissolve with mixing. Solution may turn cloudy
(indicating high level of polysaccharide in solution).
Option: if precipitate does not resuspend, use of a column may produce a low yield.
Alternatively, omit steps 9-13 and pellet nucleic acids by spinning at 15k rcf for 10min.
Wash with 70% EtOH, followed by 5min spin. Final step: resuspend in 100µl H2O.
9.
Transfer to ES RNA column and spin 30s, ≥ 10K rcf. Discard flow-through.
Note: if working with volumes greater than those suggested, transfer a portion of the
solution to the column, spin, discard the solution and repeat with remaining solution.
Note: solutions high in polysaccharide may clog the column. In this case, spin again at
higher speed.
Note: some pigment may be retained by column through following steps.
Note: if not handled with care when decanting, solutions may run down the outside of
the collection tube, contaminating gloves and work area. To prevent this, while the tube
is still inverted, lightly touch the outer-rim to a clean KimWipe placed on the lab bench.
10. Add 500ul Wash Buffer, spin 30s, ≥ 10K rcf; discard flow-through.
11. Add 500ul Wash Buffer, spin 1min, ≥ 10K rcf; discard flow-through.
12. Spin 2min ≥ 10K rcf w/empty (optional: new) collection tube.
13. Transfer column to 1.5ml tube; add 65ul H2O (RNase-free) to membrane; rest 2-5min. spin
1min ≥ 10K rcf.
Note: in some cases, H2O may be slow to enter the membrane. Lightly scrape the
membrane with pipette tip to facilitate absorption.
3
Solutions, Materials, and Recipes
Note: total cost is ~ ½ of most commercial kits
buffer f2:
2% CTAB
100mM Tris HCl pH 8.0
2% PVP40
20mM EDTA pH 8.0
2M NaCl
Wash Buffer (pH 7.4):
100mM NaCl
10mM Tris pH 7.4
70% EtOH
2ml tubes: 2.0 ml Tough Tubes with Caps (Mo Bio Laboratories, Carlsbad, CA.; cat # 13119-500)
Option: We recommend using Tough Tubes from Mo Bio. Other 2ml tubes may be used
but they may have a 5-20% failure rate when run on the FastPrep machine (Step 1 of
protocol). The brief thaw on ice can reduce the failure rate to 0.5-5%. Fisher prod. # 02681-375 (capped tubes). Non-sterile tubes from multiple manufacturers were tested and
found to be less durable when frozen tubes were processed by FastPrep. Autoclaving
these tubes (gravity cycle) improved performance. MP Bio FastPrep tubes: 115076200;
caps: 115067002. Fisher non-sterile tubes: 02-681-344; caps: 02-681-364.
Beads: ¼” steel shot: Marksman; Daisy.
Note: ¼” ceramic beads (MP Bio: 116540424) may be used but are more expensive.
FastPrep®: MP Biomedicals, Inc.
1.5ml tubes: RNse-free, DNase-free tubes of choice.
ES RNA column: Epoch Life Science, EconoSpin™ RNA column (#1940-250)
Note: possible to substitute columns from other RNA or DNA kits (e.g. RNeasy, DNeasy)
but it is important to confirm that columns will efficiently bind DNA (while some Zymo
Research columns will bind RNA and DNA, others are designed for RNA only).
Note: some columns (such as Zymo Research IIC) are more likely to clog with solutions
high in polysaccharide. Faster/longer spin times may be necessary.
optional: spare collection tubes: Epoch Life Science.
f2 recipe:
10% CTAB
1M Tris HCl pH 8.0
20% PVP40
0.5M EDTA pH 8.0
4M NaCl
H2O to 50ml
10ml
5ml
5ml
2ml
25ml
3ml
Note: Prepare 50ml stock of 10% CTAB; necessary to stir overnight or heat and
stir to get CTAB into solution. Once it is prepared, store it in a bottle with stir
4
bar (CTAB will precipitate over time). To resuspend CTAB, stir on hot/stir plate
with heat set to lowest setting for about ½ hour before needed.
Note: Prepare 50ml stock of 20% PVP in 50ml tube. No shaking is needed; it will
take ~2 days for PVP to go into solution and it will stay in solution.
3M KOAc (pH 5.2, recipe from Sambrook/Maniatis)
5M KOAc
H2O
acetic acid
60ml
28.5ml
11.5ml
Note: actual pH may vary from 5.2 to 5.5; obtaining a pH of exactly 5.2 is not necessary.
Wash Buffer recipe (RNase free): 50ml
H2O
13.5ml
1M Tris pH 7.4
0.5ml
5M NaCl
1ml
100% EtOH
35ml
RNase-free buffers, salts, and H2O available from Ambion (Applied Biosystems).
1M Tris pH 7.4 (7.39) RNase free (Ambion recipe): 10ml
1M Tris pH 7
8ml
1M Tris pH 8
2ml
RNase-free buffers and H2O available from Ambion (Applied Biosystems).
references:
R. Li, R. Mock, Q. Huang, J. Abad, J. Hartung, and G. Kinard, 2008 A reliable and inexpensive method of nucleic acid
extraction for the PCR-based detection of diverse plant pathogens. J. of Virological Methods 154, 48-55.
I. Echevarría-Machado, L.A. Sánchez-Cach, C. Hernández-Zepeda, R. Rivera-Madrid, and O.A. Moreno-Valenzuela, 2005,
A simple and efficient method for isolation of dna in high mucilaginous plant tissues. Molecular Biotechnology 31, 129135.
Sambrook, J., E. F. Fritsch, et al. (1989). Molecular cloning : a laboratory manual. Cold Spring Harbor, N.Y., Cold Spring
Harbor Laboratory.
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