BP-10 Spin Column Genomic DNA Minipreps Kit, Bacteria

a)DigestionSolution may form a precipitate upon storage. If necessary, dissolve theprecipitate by warming the solution at 37ºC.

b)Before use, add96ml of 100% ethanol to 24ml Wash Solution. For other volumes of WashSolution, simply add enough ethanol to make a 4:1 ratio (volume of addedethanol : volume of Wash Solution = 4:1).

c)Elution Buffer is2.0mM Tris-HCl pH 8.0-8.5. Although Tris buffer pH 8.0 or water can be used, yield may beslightly lower.

d)Before use, add300µl of sterilized water to the tube containing 4mg of proteinase K. For long termstorage store proteinase K solution at -20ºC.

Principle

This kit is designed forfast isolation of genomic DNA fromcells and bacteria. The kit contains a membrane embedded column for binding upto 10µg of genomic DNA.Nucleotides, proteins, salts, and other impurities are washed away. Purifiedgenomic DNA can be used in mostmolecular biology experiments including restriction enzyme digestion, PCR,Southern-blotting, etc.

Procedures for Isolation ofGenomic DNA from Cells and Bacteria

1. Sample Preparation
   1. Cell Cultures
      1. Cells grown in suspension
         1. Spin appropriate number of cells (max. 5 million cells) at 2,500 x g (5,000 rpm) for 5 minutes at room temperature. Remove supernatant completely and discard. Wash the cell pellet twice with PBS and resuspend cells in 200µl cold TE, proceed to Step 2.
      2. Cells grown in monolayer
         1. Aspirate the medium and wash cells with PBS. Remove PBS and apply trypsin solution to the cells. After cells have become detached, neutralize the trypsin with 2 volumes of medium. Centrifuge at 8,000 x g (10,000 rpm) for 5 minutes. Carefully remove supernatant and resuspend pellet in 200µl TE buffer, and continue immediately with Step 2.
      3. Notes
         1. If sample willnot be used immediately for genomic DNAextraction, it is recommended to store at -20ºC or -80ºC for long-term.
         2. Avoid repeatedfreezing and thawing of stored samples, since this leads to reduced DNA size and yield.
   2. Bacteria Collection
      1. Spin appropriate number of bacteria (about 106~107) at 6,000 x g (8,000 rpm) for 5 minutes at room temperature. Remove supernatant completely and discard, then resuspend cells in 200µl cold TE, proceed to Step 2.
   3. Paraffin Tissue
      1. Excise 25~30mg paraffin tissue with a clean, sharp scalpel. Transfer to a 1.5ml Eppendorf tube
      2. Add 1.2ml xylene (not included in the kit) to the tube, then vortex for 3 minutes. Xylene is used to remove paraffin.
      3. Centrifuge at 10,000 x g (12,000 rpm) for 5 minutes at room temperature.
      4. Remove the supernatant completely. Keep the pellet.
      5. Add 1.2ml of 100% ethanol to the tube. Gently vortex for 1 minute. Incubate at room temperature for 1 minute.
      6. Centrifuge at 10,000 x g (12,000 rpm) for 5 minutes at room temperature. Discard supernatant completely.
      7. Repeat step 5 to 6.
      8. Incubate at 37ºC for 10-15 minutes to remove residual ethanol.
      9. Resuspend the sample in 200µl TE buffer, and proceed to Step 2.
2. Add 400µl of Digestion Solution to 200µl sample from step 1. Mix well. Add 3µl Proteinase K solution (2mg/150µl), and incubate at 55ºC for 5 minutes.
   1. Notes:
      1. Do not add proteinase K solution to Digestion Solution.
      2. Incubation period depends on the nature of sample. For cell cultures, 5 minutes is generally enough to obtain complete lysate. For tissue samples it requires 3-5 hours. Longer period incubation even overnight incubation will not affect the result.
3. Add 260ml of 100% ethanol, and mix well.Apply the mixture onto an EZ-10 spin column that is placed in a 2.0ml Collection Tube. Spin at 8,000 x g (10,000 rpm) for 2 minutes.
4. Discard the flow-through in the collection tube. Add 500ml of Wash Solution, and spin at 8,000 x g (10,000 rpm) for 2 minutes.
5. Repeat Step 4.
6. Discard flow-through. Spin at 8,000 x g (10,000 rpm) for an additional minute to remove residual amount of Wash Solution.
7. Place the EZ-10 column into a clean 1.5ml Eppendorf tube. Add 30-50µl Elution Buffer into the center part of membrane in the column. Incubate the tube at 37 or 50ºC for 2 minutes. Incubate at 37 or 50ºC could increase recovery yield.
8. Spin at 8,000 x g (10,000 rpm) for 2 minute to elute DNA from the column.
9. For long term storage, keep aliquots of purified genomic DNA at -20ºC.
10. Measure DNA quantity by UV absorption at A260 (1.0 OD unit is equivalent of 50 µg). Assess genomic DNA quality by an analytical 0.7% agarose gel. Isolated genomic DNA should not contain RNA. Its length should be over 50 kb.

Troubleshooting Guide: BP-10 Spin Column Genomic DNAMinipreps Kit, Bacteria

1. Low yield
   1. Improper storage of startingmaterial
      1. Prepare fresh samples and use immediately.
   2. Too much or too LOW starting material
      1. Reduce or increase starting material accordingly.
   3. Incorrect preparation of buffers
      1. Each step has to be strictly followed.
2. RNA contamination
   1. Perform optional RNasetreatment according to the protocol.
3. OD 260nm/280nm ratio outside 1.9-2.2 range
   1. If the ratio of OD260nm/280nmis greater than 2.2, there may be traces of ethanol present. If the ratio ofOD260nm/280nm is smaller than 1.9, there may be chance of proteincontamination. Make sure the sample is mixed well after proteinase K digestion.
4. 4. DNA does not perform well
   1. DNA Shearing
      1. Avoid repeated freezing and thawing of starting material; if samples are too old, start with a new sample.
   2. thanol Carryover
   1. Spin additional minute before elution