Carnegie Institution 
of Washington, DPB

 

Supplemental material for:

Positional cloning in Arabidopsis -
why it feels good to have a Genome Initiative working for you.
 

Plant Physiology, July 2000, Vol. 123(3)
 

Wolfgang Lukowitz, C. Stewart Gillmor and Wolf-Rüdiger Scheible
Carnegie Institution of Washington, Department of Plant Biology,260 Panama Street, Stanford, CA 94305, U.S.A.
Email: lubo/gillmor/scheible@andrew2.stanford.edu

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PREPARATION OF ARABIDOPSIS DNA FOR PCR PURPOSES

Protocol 1: CTAB extraction

Modified by Farhah Assaad (unpublished) after Murray MG, Thompson WF (1980) Rapid isolation of high-molecular-weight plant DNA.  Nucl Acid Res 8: 4321-4325 (Abstract).

Reagents:
 

2X CTAB buffer:

2% (w/v) cetyl-trimethyl-ammonium bromide (CTAB)
1.4 M NaCl
100 mM Tris HCl pH 8.0
20 mM EDTA

Chloroform

2-Propanol

70% Ethanol

TE buffer (10 mM Tris HCl pH 8.0, 1 mM EDTA)

Procedure:

1. Mash or grind a small amount of plant tissue in a 1.5 ml reaction tube
2. Add 300 ml 2X CTAB buffer and incubate at 65 oC for at least 10 minutes (up to several hours)
3. Allow to cool
4. Add 300 ml chloroform and vortex thoroughly
5. Spin briefly in a microfuge to separate phases
6. Transfer the upper, aqueous phase to a fresh reaction tube
7. Add 300 ml 2-Propanol and mix well
8. Spin in a microfuge for 5 minutes to pellet the DNA
9. Remove supernatant and wash pellet with 500 ml 70% ethanol
10. Spin briefly in a microfuge
11. Carefully remove the ethanol and air dry pellet
12. Add 100 ml TE buffer and allow the pellet to dissolve
13. Mix or vortex before use. Use 1-2 ml in a PCR reaction

 


Notes:
 

 

 

A.  This protocol yields relatively clean, high molecular weight DNA (see image of agarose gel). We have successfully used this DNA for Southern blotting and PCR amplification of long DNA fragments (up to 10 kilobase pairs).

B.  A single rosette leaf or an inflorescence provide sufficient starting material. The tissue can be used fresh or stored at ?20o C prior to processing. If more than 100 mg of tissue is used, the amount of buffer should be adjusted such that it equals roughly four times the amount of tissue.

C.  Plastic pestles that fit 1.5 ml reaction tubes can be used to grind the tissue (step 1). Appropriate pestles can be purchased from the Kimble-Kontes Glass Company (Vineland, NJ; www.kimble-kontes.com; catalog# 749521-1500). In this case, the tissue should be ground with the 2X CTAB buffer added. The pestles can be mounted in a power drill for easier grinding. If only co-dominant markers are analyzed, it is not necessary to use a fresh pestle for each tissue sample - briefly rinsing the pestle in water will be sufficient to avoid visible cross contamination. As a cheap alternative to pestles, 1 ml pipettor tips can be used to mash the tissue. In this case, the tissue should be mashed without buffer by pressing against the walls of the tube until only small fragments remain.

D. When transferring the aqueous phase (step 6), it is often difficult to avoid carry-over of some material accumulating at the interphase between chloroform and buffer. In our experience, this contamination will not affect PCR reactions.

E.  Upon precipitation, a pellet should be visible (step 8).

F. The DNA can be stored at 4 oC for several weeks or at -20 oC for a prolonged time.
 

 

Agarose gel electrophoresis of CTAB DNA preparations from a single Arabidopsis inflorescence (lane 1) and a single rosette leaf (lane 2). One tenth of each DNA preparation was loaded. A standard (l DNA/BstE II digest,500 ng total DNA) is shown in lane 3

X
X


 

Protocol 2: Alkaline lysis

After Klimyuk VI, Carroll BJ, Thomas CM, Jones JDG (1993) Alkali treatment for rapid preparation of plant material for reliable PCR analysis. Technical Advance. Plant J 3: 493-494 (Abstract).

Reagents:

0.25 N sodium hydroxide

0.25 N hydrochloric acid

0.5 M Tris HCl pH 8.0 / 0.25% (v/v) IGEPAL CA-630

Procedure:

1. Mash tissue in 50 ml 0.25 N sodium hydroxide
2. Incubate for 30 seconds at 96 oC
3. Add 50 ml 0.25 N hydrochloric acid and 25 ml 0.5 M Tris HCl pH 8.0 / 0.25% (v/v) IGEPAL CA-630
4. Incubate for 2 minutes at 96 oC
5. Use 1-2 ml of the crude lysate in a PCR reaction

Notes:

A. The crude lysate will allow PCR amplification of small DNA fragments, such as SSLP markers. However, we have occasionally experienced problems with the amplification of larger fragments (500 base pairs and longer). It is advisable to determine before-hand if a given marker can reliably be amplified from this kind of template.

 


B. Part of a young rosette leaf or a small inflorescence (about 5 buds) provides sufficient starting material. The tissue material can be used fresh or stored at -20 oC prior to processing. Processing significantly larger amounts of tissue may lead to problems with PCR amplification due to the presence of inhibiting substances in the lysate.

C. Typically, this protocol is used to process a large number of tissue samples in a microtiter plate format. A 96-well plate for use in a thermocycler is ideal for the purpose. The plate can be placed on ice in a rack for 10m l pipettor tips for better support. Using the round end of flexible polyethylene stirring rods that exactly fit into the wells of the microtiter plate, several tissue samples can be mashed simultaneously (step 1; see images). Appropriate stirring rods can be purchased from Sarstedt (Nümbrecht, Germany; www.sarstedt.com; catalog# 81-970). The tissue is sufficiently mashed if the sodium hydroxide solution turns bright green or yellow-green from the extracted pigments. Heating can conveniently be done in a thermocycler (steps 2 and 4). To avoid cross-contamination of neighboring samples, microtiter plates should not be covered with lids or tape during the heating steps.

D. The crude lysate can be stored at -20 oC for several weeks.

 

 

 

 

 

 

A COLLECTION OF 22 SSLP MARKERS FOR BULKED SEGREGANT ANALYSIS

The table below contains technical information on 22 SSLP markers that are spaced at a distance of 10 to 30 % recombination over the entire Arabidopsis genome and can be used for bulked segregant mapping. The nga markers, F21M12, CTR1 and PHYC have been described previously (Bell CJ, Ecker JR (1994) Assignment of 30 microsatelite loci to the linkage map of Arabidopsis. Genomics 19: 137-144 (Abstract); http://genome.bio.upenn.edu/SSLP_info/SSLP.html), the ciw markers were created by C. S. G.
 


 

Chr.

(cM)

Marker

(BAC)

Forward primer (5’->3’)

Reverse primer (5’->3’)

Col

Ler

(bp)

Ws

[MgCl2]

(mM)

I

(10)

F21M12

(F21M12)

ggctttctcgaaatctgtcc

ttactttttgcctcttgtcattg

200

~160

~215

2.0

(39)

ciw12

(T22C5)

aggttttattgcttttcaca 

ctttcaaaagcacatcaca

128

~115

~113

1.5

(72)

ciw1

(F14J22)

acattttctcaatccttactc 

gagagcttctttatttgtgat

159

~135

~130

2.0

(81)

nga280

(F14J16)

ctgatctcacggacaatagtgc 

ggctccataaaaagtgcacc

105

85

85

1.5

(113)

nga111

(F28P22)

 tgttttttaggacaaatggcg

ctccagttggaagctaaaggg

128

162

146

1.5

II

(11)

ciw2

(T18C20)

cccaaaagttaattatactgt 

ccgggttaataataaatgt

105

~90

~105

2.5

(30)

ciw3

(T26I20)

gaaactcaatgaaatccactt 

tgaacttgttgtgagctttga

230

~200

~1000

2.5

(50)

nga1126

(F10A12)

cgctacgcttttcggtaaag 

gcacagtccaagtcacaacc

191

199

191

2.0

(73)

nga168

(T7F6)

tcgtctactgcactgccg 

gaggacatgtataggagcctcg

151

135

135

2.0

III

(20)

nga162

(MDC16)

catgcaatttgcatctgagg 

ctctgtcactcttttcctctgg

107

89

85

1.0

(43)

ciw11

(MFE16)

ccccgagttgaggtatt 

gaagaaattcctaaagcattc

179

~230

~240

2.5

(70)

ciw4

(F18B3)

gttcattaaacttgcgtgtgt 

tacggtcagattgagtgattc

190

~215

~190

2.5

(86)

nga6

(T17J13)

tggatttcttcctctcttcac 

atggagaagcttacactgatc

143

123

131

1.0

IV

(10)

ciw5

(T15B16)

ggttaaaaattagggttacga 

agatttacgtggaagcaat

164

~144

~500

2.0

(47)

ciw6

(T6G15)

ctcgtagtgcactttcatca 

cacatggttagggaaacaata

162

~148

~135

2.0

(65)

ciw7

(F17L22)

aatttggagattagctggaat 

ccatgttgatgataagcacaa

130

~123

~150

2.0

(104)

nga1107

(T9A14)

gcgaaaaaacaaaaaaatcca 

cgacgaatcgacagaattagg

150

~140

~140

1.5

V

(10)

CTR1

(F7P15)

ccacttgtttctctctctag 

tatcaacagaaacgcaccgag

159

143

145

2.5

(42)

ciw8

(MQJ16)

tagtgaaacctttctcagat 

ttatgttttcttcaatcagtt

100

~135

~100

2.0

(71)

PHYC

(MIK22)

ctcagagaattcccagaaaaatct

aaactcgagagttttgtctagatc

207

222

222

2.0

(88)

ciw9

(MFO20)

cagacgtatcaaatgacaaatg 

gactactgctcaaactattcgg

165

~145

~145

1.0

(115)

ciw10

(MSL3)

ccacattttccttctttcata 

caacatttagcaaatcaactt

140

~130

~138

2.0

NB: On October 19th, 2000, the sizes of several Ws markers were revised


All markers can be amplified using this PCR profile:
(thermocycler model PTC-100 from MJ Research)


1 minute @ 94 oC; 

40 cycles: 
30 seconds @ 94 oC, 
30 seconds @ 55 oC, 
30 seconds @ 72 oC

 


The concentrations of reagents in the PCR reaction are:

buffer (50 mM potasium chloride, 10 mM Tris-HCl pH 9.0 @ room temperature, 0.1% Triton X-100)

200 mM dNTPs (each)

1 mM primer (each)

1.0 to 2.5 mM MgCl2 (see table)

template DNA (2 ml of a CTAB preparation per 20 ml reaction)

0.5 U Taq polymerase (per 20 ml reaction)
 

A convenient way to set-up the PCR reactions is as follows: For each of the 22 markers, 4X stock solutions containing both primers and magnesium are prepared (4 mM of each primer and, dependent on the primer pair, 4, 6, 8 or 10 mM MgCl2). For both DNA samples, the pooled mutant DNA and the heterozygous control DNA, a master mix containing all the other reagents (buffer, dNTPs, template DNA and enzyme) is prepared. Aliquots of the primer / MgCl2 stocks are distributed on a microtiter plate and the master mix is added.

The PCR products are analyzed on high-resolution agarose gels in order to resolve the small size differences. We use 4% gels prepared with a high-resolution agarose blend (agarose 3:1) from Amresco (Solon, OH; www.amresco-inc.com; catalog# E776) and 1X TBE as running buffer (3 Vcm-1 for 3-5 hours).
 
 

click here to download a print-version (PDF file) of this page

 

Carnegie Institution 
of Washington,DPB