Isolation of high molecular weight DNA suitable for BAC library construction from woody perennial soft-fruit species.
We have developed a novel nuclei extraction method that allows for the extraction of high molecular weight DNA from leaves of woody perennial soft-fruit species that contain high levels of carbohydrates and polyphenolics. The method utilizes a modified buffer system including 4% (w/v) polyvinylpyrrolidone (PVP)-10 and a combination of nylon filters and Percoll gradients to purify nuclei extracts prior to embedding in agarose plugs. The effectiveness of the method was demonstrated on leaves of red raspberry (Rubus idaeus) and blackcurrant (Ribes nigrum), two soft-fruit species that have shown to be recalcitrant to standard genomic DNA extraction methods. Extracted DNA was readily digested by restriction enzymes and, as shown for raspberry, suitable for bacterial artificial chromosome (BAC) library construction.
Hein
Ingo
I
Scottish Crop Research Institute, Programme of Genome Dynamics, Invergowrie, Dundee, UK. ihein@scri.sari.ac.uk
Williamson
Sandie
S
Russell
Joanne
J
Powell
Wayne
W
D022202:Chromosomes, Artificial, Bacterial / Q000235:genetics*; D018744:DNA, Plant / Q000737:chemistry* / Q000302:isolation & purification*; D005590:Fractional Precipitation; D015723:Gene Library; D008970:Molecular Weight; D018515:Plant Leaves / Q000235:genetics*; D019685:Rosales / Q000235:genetics*; D013048:Specimen Handling / Q000379:methods; D014462:Ultrafiltration / Q000379:methods*
D018744:DNA, Plant