Advanced Search Opt.
Read our latest customer feedback on successful plant DNA extrac
Who says Plant DNA extractions are difficult?.... Just try the Macherey-Nagel Nucleospin Kits from D-Mark Bio and you’ll never look back!
We are pleased to share with you some technical tips from our customers, courtesy of the Aitkin Lab at the UBC Forestry Department, who have been collecting field samples of Lodgepole Pine and Interior Spruce as part of their AdapTree program. They have some great recommendations for dealing with the DNA extractions from these difficult sample types, using the Nucleospin Plant Kits. They worked in a 96-well format, with vacuum, but their recommendations would be applicable for both the Nucleospin Plant single-prep columns and plate-based extractions with the Nucleospin 96 Plant. Some highlights include:
· The Nucleospin Plant kits come equipped with 2 standard protocols and two lysis buffer options, PL1 (CTAB-based lysis buffer) or PL2 (SDS-based lysis buffer). The Aitkin lab determined that the PL2 SDS-based buffer worked better for both Pine and Spruce needles, and that the pre-column protein clearing step in the PL2 protocol greatly increased DNA yield and purity. This is contrary to the accepted notion that CTAB is the “go-to” extraction method for difficult plant samples. CTAB has been used for years on “woody” tissues.
· 20mg of tissue (dried) also seemed to be best, with less not giving enough DNA, and more input also decreasing in yield. 20mg of dry tissue is the maximum recommended input for this kit – The results show that the column was saturated with DNA at this input, indicating good lysis. Note: If processing of larger inputs is desired, these same lysis buffers/reagents can be combined with a more scalable bead-based extraction (i.e. NucleoMag Plant kits)
· It was also noted that increasing the lysis incubation to 45 minutes at 65C and increasing the protein precipitation to 20 minutes at -20C also helped to improve DNA yield and purity from these tough samples.
· Another modification was the addition of PVPP (polyvinylpolypyrrolidone) to the PL2 Lysis buffer, which inactivates phenolic and polysaccharide compounds in the mixture, improving DNA recovery. Addition of 1% PVPP improved the average 260/230 from 1.75 to 2.33, and increased yield and reproducibility.
See the link below to read their blog post: http://blogs.ubc.ca/aitkenlab/2014/04/02/lodgepole-pine-dna-extractions/