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Table 1 Overview of nuclei isolation protocols A, B and C

From: Isolation and analysis of high quality nuclear DNA with reduced organellar DNA for plant genome sequencing and resequencing


Protocol A

Protocol B

Protocol C

Plant criteria

•Low amount of plant material available

•Low levels of secondary metabolites

•DNase-rich plants

•Large amounts of plant tissue available

•High levels of secondary metabolites

Successful DNA isolation

A. thaliana

B. distachyon

G. aurea

S. bicolor

L. gibba*

S. polyrhiza*

Z. mays

B. distachyon

L. gibba

S. bicolor

S. polyrhiza

Z. mays

•V. macrocarpon

Protocol Modifications

•Used only 0.25 g tissue

•Only performed DNA isolation steps (6-13)

•Removed protease inhibitors from all buffers

•Resuspended nuclei pellet in TE (10 mM:1 mM)

•Omitted TE slurry and diethyl ether steps

•Increased volume of SEB to 200 ml

•Added Triton X-100 drop by drop to minimize disruption of the nuclear membrane

•Isolated DNA by isopropanol precipitation

•Omitted TE slurry and diethyl ether steps

•Added EGTA and L-Lysine-HCl to MEB Buffer

•Incubated sample on ice 8 minutes after addition of Triton X-100

•Centrifuged filtrate one time

•Resuspended nuclei pellet in 1 ml of MPDB per gram of starting tissue

Protocol modified from

Gendrel et. al. [15]

Peterson et. al. [16] (Option Y)

Peterson et. al. [16] (Option X); Peterson et. al. [17]

  1. * For L. gibba and S. polyrhiza the volume of buffer A had to be doubled for isolation on non-degraded DNA.