BP-10 Spin Column Genomic DNA Isolation Kit (For Animal)

Kit Contents:

A.Before use, add 1ml or 2ml of sterilized water to the tube containing 20mg or 40mg of proteinase K, keep at -20 ^oC.

B.Before use, add 48ml of 100% ethanol to 12ml Wash Solution for GDMIP-50, add 96ml of 100% ethanol to 24ml Wash Solution for GDMIP-100. For other volumes of wash solution, simply add enough ethanol to make a 4:1 ratio (volume of added ethanol : volume of Wash Solution = 4:1).

C.Elution Buffer is 2.0 mM Tris-HCl pH 8.0-8.5. Although TE buffer pH 8.0 or water can be used, yield is generally 10% lower.

Storage:

With the exception of the Proteinase K, the kit may be stored at room temperature. The proteinase K should be stored at 4ºC. The kit is stable for 12 months at room temperature. For longer storage, keep all contents cold.

Principle:

This kit is designed for fast isolation of genomic DNA from animal tissues. The kit contains a membrane embedded in BP-10 spin column for binding up to 10ug of genomic DNA. Nucleotides, proteins, salts, and other impurities do not bind to the column. Purified genomic DNA can be applied in most molecular biology experiments including restriction digestion, PCR, Southern-blotting etc.

Applications:

Genomic DNA purification from different animal tissues.

Features:

• Preparation of high quality genomic DNA from variable sources.
• Rapid and economical.
• High yields
• No phenol / chloroform extraction , no ethanol precipitation

Procedure for Isolation of Genomic DNA from Variable Sources.

For Animal Tissue

1. Cut up to 30mg tissue and place in a 1.5ml centrifuge tube.
2. Add 300ul of ACL Solution (Animal Cell Lysis Solution) to 1.5ml centrifuge tubes and 20ul proteinase K.
3. Incubate at 55^oC until the tissue is completely lysed (usually 1-3 hours). Occasionally vortex. Incubation in shaking water bath can reduce lysis time.
4. Cool to room temperature. Vortex for 20 seconds and Centrifuge 12000 rpm for 5 minutes.
5. Pipette 300ul of supernatant into an BP-10 spin column (if pellet not visible repeat previous step) and add 300ul of AB Solution . Mix by occasionally inverting tube and keep for 2 minutes.
6. Centrifuge 4000 rpm for 2 minutes and discard the flow-through.
7. Add 500ul of Wash Solution, and spin at 8,000 rpm for 1 minute.
8. Repeat washing step 7.
9. Discard flow-through. Spin at 10,000 rpm for an additional minute to remove residual amount of Wash Solution.
10. Place the column into a clean 1.5 ml centrifuge tube. Add 30-50ul Elution Buffer into the center part of membrane in the column. Incubate the tube at 37 or 50^oC for 2 minutes. Incubate at 37 or 50^oC could increase recovery yield.
11. Spin at 10,000 rpm for 1 minute to elute DNA from the column.
12. Measure DNA quantity by UV absorption at A260 (1.0 OD unit is equivalent of 50 ug). 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.

For Rodent Tail

1. Place numbered 1.5ml centrifuge tubes in dry ice.
2. Cut 0.5cm to 1cm from end of tails and place in tubes.
3. Add 300ul of ACL Solution to 1.5ml centrifuge tubes and 20ul proteinase K.
4. Incubate at 55^oC overnight with rocking or for several hours with occasional mild vortexing every 15 minutes.
5. Cool to room temperature. Vortex 20 seconds and Centrifuge 12000 rpm for 5 minutes.
6. Pipette 300ul of supernatant into an BP-10 spin column (if pellet not visible , repeat previous step) and add 300ul AB Solution . Mix by occasionally inverting tube and keep for 2 minutes.
7. Centrifuge 4000 rpm for 2 minutes and discard the flow-through.
8. Add 500ul of Wash Solution, and spin at 8,000 rpm for 1 minute.
9. Repeat washing step 8
10. Discard flow-through. Spin at 10,000 rpm for an additional minute to remove residual amount of Wash Solution.
11. Place the column into a clean 1.5ml centrifuge tube. Add 30-50 ul Elution Buffer into the center part of membrane in the column. Incubate the tube at 37 or 50^oC for 2 minutes. Incubate at 37 or 50oC could increase recovery yield.
12. Spin at 10,000 rpm for 1 minute to elute DNA from the column.
13. Measure DNA quantity by UV absorption at A260 (1.0 OD unit is equivalent of 50 ug). 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.

For Cultured Animal Cell

1. Centrifuge the appropriate number of cells(>5x10⁶) for 5 minutes at 1200 rpm.
2. Resuspend pellet in 200ul of PBS Solution.
3. Add 20ul of proteinase K .
4. Incubate at 70^oC for 10 minutes.
5. Cool to room temperature. Vortex for 20 seconds and Centrifuge 12000 rpm for 5 minutes.
6. Pipette 200ul of supernatant into an BP-10 spin column (if pellet not visible , repeat previous step) and add 200ul AB Solution. Mix by occasionally inverting tube and keep for 2 minutes.
7. Centrifuge 4000 rpm for 2 minutes and discard the flow-through.
8. Add 500ul of Wash Solution, and spin at 8,000 rpm for 1 minute.
9. Repeat washing step 8
10. Discard flow-through. Spin at 10,000 rpm for an additional minute to remove residual amount of Wash Solution.
11. Place the column into a clean 1.5ml centrifuge tube. Add 30-50ul Elution Buffer into the center part of membrane in the column. Incubate the tube at RT for 2 minutes. To incubate at 37 or 50^oC could increase recovery yield.
12. Spin at 10,000 rpm for 1 minute to elute DNA from the column.
13. Measure DNA quantity by UV absorption at A260(1.0 OD unit is equivalent of 50ug). 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.