BP-10 Spin Column Genomic DNAMinipreps Kit, Plant

Introduction

The BP-10Spin Column Kits provide a simple and efficient method for purification ofgenomic DNA from different sources such as Bacteria, Plant, Animal and Blood.

The DNA isselectively adsorbed in silica gel-based BP-10 Spin Column and other componentsare washed away. The genomic DNA is eluted off the column and can be used forany downstream applications, including restriction enzyme digestion, PCR,Southern-blotting, etc.

Thepurification methods used in these protocols do not require use of phenol,chloroform, or CsCl. The DNA is purified without an additional step of ethanolprecipitation.

Limitations of Use

These kitsare designed for research only. The purified DNA should not be used for liveanimal transfections. It is also not to be used for human diagnostic or drugproduction purposes.

Features

• Simple, fast and efficient
• Preparation of high quality genomicDNA from various sources
• High yield and reproducible
• No phenol / chloroform extraction orethanol precipitation required
• High capacity: up to 10µg of DNA percolumn

Applications

Efficient ofup to 10µg of genomic DNA purification from different sources

Storage

With theexception of the Proteinase K, the kit may be stored at room temperature.Proteinase K should be stored at 4ºC for short term or -20ºC for longterm. The kit is stable for 12 months at room temperature. For longer storage,keep all contents in cold place.

Quality Control

Each lot ofBP-10 Spin Column kit is tested against predetermined specifications to ensureconsistent product quality.

• PCLSolution DOES NOT contain RNase A (100 µg/ml). Please add entire contents ofRNAse A into PCL Solution and store at 4ºC for long term storage. PCL Solution may form a precipitate upon storage. If necessary,dissolve the precipitate by warming up to room temperature.
• Beforeuse, add 96ml of 100% ethanol to 24ml Wash Solution for GDMIP-100P.For other volumes of wash solution, simplyadd enough ethanol to make a 4:1 ratio (volume of added ethanol : volume of WashSolution = 4:1).
• ElutionBuffer is 2.0mM Tris-HCl pH 8.0~8.5. Although TE buffer pH 8.0 or water can beused, yield may be 20% lower.

Procedures for Isolation of Genomic DNA from Plants

1. PlantTissue Sample Preparation
   1. Grind plant tissue under liquid nitrogen to a finepowder using a mortar and pestle. Transfer the powder and liquid nitrogen to1.5ml Eppendorf tube and allow liquid nitrogen to be evaporated. Do not allowthe sample to thaw. Proceed immediately to Step 2.
   1. Note:
   1. Ifsample can not be used immediately for genomic DNA extraction, it isrecommended to store at -20ºC for long-term use.
   2. Avoid repeated freezing and thawing of stored samples, since this leads to reduced DNA size and yield.
   3. Incubation period depends on the nature of sample. Longer period incubation even overnight incubation will not affect the result.
   4. Better results could be achieved if starting material is less than 60mg.In any case, the quantity of samples should not exceed 100 mg for one BP-10 Spin Column.
2. Checkthe volume of grounded material, and add equal volume (approximately 150µl) ofPCL Solution (Plant Cell Lysis Solution).
3. Vortexand shake the tube several times. Incubate at 65ºC for 20 minutes,vortex or pipette up and down to further remove any clumps. Clumped tissue willnot lyse properly and will result in a lower yield of DNA.
4. Add25µl PP Solution. Mix well. Keep the solution on ice for 15 minutes.
5. Centrifugeat 4ºC, 8,000 x g (10,000 rpm) for 5 minutes. Apply the clearlysate to an BP-10 Spin Column.
6. Add300µl PB buffer to the BP-10 Spin Column. Mix gently by inverting the tube.Incubate the mixture for 3 minutes at room temperature. During incubation, mixoccasionally by inverting the tube.
7. Centrifugeat 4ºC, 8,000 x g(10,000 rpm) for 2 minutes.
8. Discardthe flow-through in the Collection Tube. Add 500µl of Wash Solution, and spinat 8,000 x g (10,000 rpm) for 2 minutes.
9. RepeatStep 8.
10. Discard flow-through. Spin at 8,000 xg (10,000 rpm) for an additional minute to remove residual amount of WashSolution.
11. Place the column into a clean 1.5mlEppendorf tube. Add 30-50µl Elution Buffer into the center part of membrane inthe column. Incubate at RT for 2 or 3 minutes. Incubating the tube at 37 or 50ºC for 2 minutes may increase recovery yield.
12. Spin at 8,000 x g (10,000 rpm) for 2minutes to elute DNA from the column. Measure DNA quantity by UV absorption atA₂₆₀ (1.0 OD unit is equivalent of 50µg).
13. For long term storage, keep aliquotsof purified genomic DNA at -20ºC.

Measure DNA quantity by UV absorption at A₂₆₀(1.0 OD unit is equivalent of 50 µg). Assess genomic DNA quality by ananalytical 0.7% agarose gel. Isolated genomic DNA should not contain RNA. Itslength should be over 50 kb.

Troubleshooting Guide: BP-10 SpinColumn Genomic DNAMinipreps Kit, Plant

1. Low Yield
   1. There are a number of variables that can cause low yield.
   1. Inefficienthomogenization.
      1. Ensure that the material is completely disrupted. If necessary, increase time of homogenization.
   2. Low DNA content of plant tissue.
   1. Increase the amount of starting material to 200mg.
2. RNA contamination
   1. RNase activity is weakened or lost.Add 30% additional RNAse A, and store solution at 4ºC.
3. OD 260nm/280nm ratio outside1.9-2.2 range
   1. If the ratio of OD260nm/280nm is greater than 2.2, there maybe traces of ethanol present. If the ratio of OD260nm/280nm is smaller than1.9, there is a chance of protein contamination. Make sure the sample is mixedwell after Proteinase K digestion.