DNA preparation

 

CTAB-DNA preparation for individual samples

This method is used to prepare genomic DNA of A. thaliana from individual samples. We use this DNA for generating confirmation amplicons to validate FST-predicted insertions.

 

- transfer 5-10 approx. 2 week old plantlets into a 1.5 ml reaction tube

- add 2 metal beads (tungsten carbide)

- homogenize with a TissueLyser (QIAGEN) for 2 x 2 min at 30/s or with any other device of your choice

- add 300 µl of CTAB-1 Puffer

- incubate for 5 min at 65-70°C (water bath or thermo mixer)

- cool for 5 min to RT (room temperature)

- add 300 µl of Dichloromethane and mix gently by inverting the reaction tube

- centrifuge for 20-40 min at full speed (table top centrifuge, RT)

- transfer upper phase into a new 1.5 ml tube

- add 600 µl CTAB-2

- incubate for 5 min at room temperature (incubation over night possible)

- centrifuge for 20 min at full speed (table top centrifuge, RT)

- discard supernatant

- add 100 µl of 1 M NaCl and re-dissolve the pellet (if the pellet does not dissolve incubation at 37 °C may be required)

- precipitate DNA by adding 100 µl of isopropanol

- incubate for 2h at room temperature (over night possible)

- centrifuge for 20 min at full speed (table top centrifuge, RT)

- discard supernatant

- wash once with 200 µl of 70-80% ethanol

- centrifuge for 10 min at full speed (table top centrifuge, RT)

- dry pellet for 2h at room temperature (over night possible)

- re-dissolve pellet in 50-100 µl of CTAB-TE at 60-65°C

 

In general, 1-2 µl of this genomic DNA solution is sufficient as template for the subsequent confirmation PCR.

 

CTAB Puffer 1

2 % CTAB

100 mM Tris-HCl, pH 8.0

20 mM EDTA

1,4 M NaCl

0.5 % PVP (optional)

add DTT before use (1:100) from a 1 M stock (optional)

 

CTAB Puffer 2

1.0 % CTAB

50 mM Tris-HCl, pH 8.0

10 mM EDTA

0.25 % PVP (optional)

 

CTAB-TE

10 mM Tris, pH 8.0

0.1 mM EDTA pH 8.0

7.5 µg/ml RNAse (0.75 ml from a 10 mg/ml stock per L)

 

 

DNA preparation in 96 well format

This mircotiter-based method is used for the preparation of genomic A. thaliana DNA, which is needed for the generation of FSTs in "block format". For details concerning generation of FSTs please check the FST generation page. We prepare DNA from leaf material using the DNeasy 96 plant Kit from QIAGEN (Hilden, Germany), according to the manufacturers’ instructions. We yield gDNA solutions with a concentration of roughly 10-100 ng/µL. Other methods which produce similarly clean gDNA should work as well.