BP-10 96-Well Plate Genomic DNA Isolation Kit (For Animal)

Kit Contents:

• ACL Solution may form a precipitate upon storage. If necessary,dissolve the precipitate by warming the solution at 37ºC.
• Before use, add 2ml of sterilized water to the tube containing 40 mg ofproteinase K, keep at -20 ^oC for long term.
• Before use, add 200 ml of 100% ethanol to 50ml Wash Solution.
• Elution Buffer is 2.0 mM Tris-HCl pH 8.0~8.5. Although TE buffer pH 8.0or water can be used, yield is generally 10% lower.

Storage: With the exception of theProteinase K, the kit may be stored at room temperature. The proteinase Kshould be stored at 4ºC for short term. The kit is stable for 18 months at roomtemperature. For longer storage, keep all contents cold.

Note: Thepurification method is based on centrifugation. There is a minimum heightrequirement of 75mm for apparatus to hold the assembly -- filter plate and deepwell plate.

Principle:

This kit is designed for fast isolation of genomicDNA from animal tissues. The kit contains a membrane embedded in BP-10 96-WellPlate for binding genomic DNA each well. Nucleotides, proteins, salts, andother impurities do not bind to the Column. Purified genomic DNA can be appliedin most molecular biology experiments including restriction digestion, PCR,Southern-blotting etc.

Applications:

Genomic DNA purificationfrom 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 30 mg tissue and place in Deep Well Collection Plate.
2. Add 300 ul of ACL Solution (Animal Cell Lysis Solution) to Deep WellCollection Plate and 20ul proteinase K, then seal.
3. Incubate at 55^oC until the tissue is completely lysed (usually 1-3 hours). Occasionallyvortex. Incubation in shaking water bath can reduce lysis time.
4. Cool to room temperature. Vortex for 20 seconds and Centrifuge 8,000rpmfor 5 minutes.
5. Pipette 300ul of supernatant into a BP-10 96-Well Plate (if pellet notvisible, repeat previous step) and add 300ul of AB Solution, Seal, Mix by occasionallyinverting Plate, and keep for 2 minutes.
6. Place a 96-Well Plate on thetop of a fresh Deep Well Collection Plate. Centrifuge at 6,000 rpm for 5 minutes witha rotor for microtiter plates.
7. Discard flow-through. Add500 ul Wash Solution to each well of 96-Well Plate and spin at 6,000 rpm for 5minutes. Discard flow-through and place BP-10 96-Well Plate back to the sameDeep Well Collection Plate.
8. Add 500 ul Wash Solution toeach well of the BP-10 96-Well Plate, spin at 6,000 rpm for 5 minutes. Discardflow-through and spin once more for 5 minutes to remove residue of WashSolution.
9. Transfer the BP-10 96-WellPlate to a 96-well storage plate. Add 30-50 ul Elution Buffer to the BP-1096-well plate; incubate at 50 ^oC for 2minutes. Centrifuge at 6,000 rpm for 5 minutes.
10. Measure DNA quantity by UV absorption at A260 (1.0 OD unit isequivalent of 50 ug). Assess genomic DNA quality by an analytical 0.7% agarosegel. Isolated genomic DNA should not contain RNA. Its length should be over 50kb.

For Rodent Tail

1. Place Deep Well Collection Plate in dry ice.
2. Cut 0.5cm to 1cm from end of tails and place in Deep Well CollectionPlate.
3. Add 300 ul of ACL Solution (Animal Cell Lysis Solution) to Deep WellCollection Plate and 20ul proteinase K, then seal.
4. Incubate at 55 ^oC overnight with rocking or for several hours with occasional mildvortexing every 15 minutes.
5. Cool to room temperature. Vortex20 seconds and Centrifuge 8,000rpm for 5 minutes.
6. Pipette 300ul of supernatant into an BP-10 96-Well Plate (if pellet notvisible, repeat previous step) and add 300ul AB Solution, Seal. Mix byoccasionally inverting Plate,and keep for 2 minutes.
7. Add 500 ul Wash Solution toeach well of the BP-10 96-Well Plate, spin at 6,000 rpm for 5 minutes. Discardflow-through.
8. Repeat washing step 7
9. Discard flow- through and spinonce more for 5 minutes to remove residue of Wash Solution.
10. Transfer the BP-10 96-WellPlate to a 96-well storage plate. Add 30-50 ul Elution Buffer to the BP-1096-well plate; incubate at 50 ^oC for 2minutes. Centrifuge at 6,000 rpm for 5 minutes.
11. Genomic DNAis ready for use or kept at –20 ^oC.

For Cultured Animal Cell (NEW!)

1. Centrifuge the appropriate number of cells(>5x10⁶) for 5minutes at 2,000rpm.
2. Resuspend pellet in 500 ul of PBS Solution.
3. Wash the cells 2 times with PBS.
4. Resuspend pellet in 300ul of ACL solution buffer.
5. Add 20ul of proteinase K.
6. Incubate at 55^oC for 10 minutes.
7. Cool to room temperature.Vortex for 20 seconds and Centrifuge 8,000rpmfor 5 minutes.
8. Pipette 200ul of supernatant into an BP-10 96-Well Plate (if pellet notvisible, repeat previous step) and add 200ul AB Solution. Mix by occasionallyinverting Plate, and keep for 2 minutes.
9. Centrifuge 6,000rpm for 5 minutes and discard theflow-through.
10. Add 500 ul of Wash Solution, and spin at 6,000 rpm for 5 minutes.
11. Repeat washing step 10
12. Discard flow- through and spin once more for 5 minutes to removeresidue of Wash Solution.
13. Transfer the BP-10 96-WellPlate to a 96-well storage plate. Add 30-50 ul Elution Buffer to the BP-1096-well plate; incubate at 50 ^oC for 2minutes. Centrifuge at 6,000 rpm for 5 minutes.
14. Genomic DNAis ready for use or kept at – 20 ^oC.
15. Measure DNA quantity by UV absorption at A260 (1.0 OD unit isequivalent of 50 ug). Assess genomic DNA quality by an analytical 0.7% agarosegel. Isolated genomic DNA should not contain RNA. Its length should be over 50kb.

FromParaffin Tissue

1. Excise 25~30mgparaffin tissue with a clean, sharp scalpel.And transfer to a DeepWell Collection Plate.
2. Add 1.2ml xylene (self-prepared by user) to Deep Well Collection Plate, Seal, and thenvortex for 3minutes.Xylene is used to remove paraffin.
3. Centrifuge at 12,000rpm for 5 minute at room temperature.
4. Remove the supernatant completely. Keep the pellet.
5. Add 1.2ml 100% of ethanol to Deep Well Collection Plate, Seal, Gently vortex for 1min. Incubate at room temperature for 1 minute.
6. Centrifuge at 8,000rpm for 5 minute at room temperature. Discard supernatantcompletely.
7. Repeat washing steponce from step4 to 6.
8. Incubateat 37 ^oC for10-15 minutes to remove residual ethanol.
9. Resuspend the sample in 200ul TE buffer, and continue immediately with Step10.
10. Add 300 ul of ACL Solution(Animal Cell Lysis Solution) to Deep Well Collection Plate and add 20ulproteinase K, then seal it.
11. Incubate at 55^oC until the tissue is completely lysed (usually1-3 hours). Occasionally vortex.
12. Cool to room temperature.Vortex for 20 seconds and Centrifuge 12000rpm for 5 minutes.
13. Pipette 300ul of supernatantinto an BP-10 96-Well Plate (if pellet not visible, repeat previous step) andadd 300ul of AB Solution, Seal. Mix by occasionally inverting Plate, andkeep for 2 minutes.
14. Centrifuge 6000rpm for 2 minutes and discard theflow-through.
15. Add 500 ul of Wash Solution,and spin at 6,000 rpm for 5 minutes.
16. Repeat washing step 15.
17. Discard flow-through. Spinat 6,000 rpm for 5 minutes to remove residual amount of Wash Solution.
18. Place the BP-10 96-WellPlate to 96-well storage plate. Add 30-50 ul Elution Buffer into the BP-10 96-WellPlate. Incubate the tube at 37 or 50 ^oC for 2minutes. Incubate at 37 or 50 ^oC couldincrease recovery yield.
19. Spin at 8,000 rpm for 5minutes to elute DNA from the column.
20. Measure DNAquantity by UV absorption at A260 (1.0 OD unit is equivalent of 50ug). Assessgenomic DNA quality by an analytical 0.7%agarose gel. Isolated genomic DNA should not contain RNA. Its length should beover 50 kb.