96 well Genomic DNA miniprep kit-Animal Tissue, 10 x96 well plates

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 of proteinase 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 8.0 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 for short term. The kit is stable for 18 months at room temperature. For longer storage, keep all contents cold.

Note: The purification method is based on centrifugation. There is a minimum height requirement of 75mm for apparatus to hold the assembly -- filter plate and deep well plate.

Principle:

This kit is designed for fast isolation of genomic DNA from animal tissues. The kit contains a membrane embedded in BP-10 96-Well Plate for binding genomic DNA each well. 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 30 mg tissue and place in Deep Well Collection Plate.
2. Add 300 ul of ACL Solution (Animal Cell Lysis Solution) to Deep Well Collection Plate and 20ul proteinase K, then seal.
3. Incubate at 55oC 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 8,000rpm for 5 minutes.
5. Pipette 300ul of supernatant into a BP-10 96-Well Plate (if pellet not visible, repeat previous step) and add 300ul of AB Solution, Seal, Mix by occasionally inverting Plate, and keep for 2 minutes.
6. Place a 96-Well Plate on the top of a fresh Deep Well Collection Plate. Centrifuge at 6,000 rpm for 5 minutes with a rotor for microtiter plates.
7. Discard flow-through. Add 500 ul Wash Solution to each well of 96-Well Plate and spin at 6,000 rpm for 5 minutes. Discard flow-through and place BP-10 96-Well Plate back to the same Deep Well Collection Plate.
8. Add 500 ul Wash Solution to each well of the BP-10 96-Well Plate, spin at 6,000 rpm for 5 minutes. Discard flow-through and spin once more for 5 minutes to remove residue of Wash Solution.
9. Transfer the BP-10 96-Well Plate to a 96-well storage plate. Add 30-50 ul Elution Buffer to the BP-10 96-well plate; incubate at 50 oC for 2 minutes. Centrifuge at 6,000 rpm for 5 minutes.
10. 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 Deep Well Collection Plate in dry ice.
2. Cut 0.5cm to 1cm from end of tails and place in Deep Well Collection Plate.
3. Add 300 ul of ACL Solution (Animal Cell Lysis Solution) to Deep Well Collection Plate and 20ul proteinase K, then seal.
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 8,000rpm for 5 minutes.
6. Pipette 300ul of supernatant into an BP-10 96-Well Plate (if pellet not visible, repeat previous step) and add 300ul AB Solution, Seal. Mix by occasionally inverting Plate,and keep for 2 minutes.
7. Add 500 ul Wash Solution to each well of the BP-10 96-Well Plate, spin at 6,000 rpm for 5 minutes. Discard flow-through.
8. Repeat washing step 7
9. Discard flow- through and spin once more for 5 minutes to remove residue of Wash Solution.
10. Transfer the BP-10 96-Well Plate to a 96-well storage plate. Add 30-50 ul Elution Buffer to the BP-10 96-well plate; incubate at 50 oC for 2 minutes. Centrifuge at 6,000 rpm for 5 minutes.
11. Genomic DNA is ready for use or kept at –20 oC.

For Cultured Animal Cell (NEW!)

1. Centrifuge the appropriate number of cells(>5x106) for 5 minutes 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 55oC for 10 minutes.
7. Cool to room temperature. Vortex for 20 seconds and Centrifuge 8,000rpm for 5 minutes.
8. Pipette 200ul of supernatant into an BP-10 96-Well Plate (if pellet not visible, repeat previous step) and add 200ul AB Solution. Mix by occasionally inverting Plate, and keep for 2 minutes.
9. Centrifuge 6,000rpm for 5 minutes and discard the flow-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 remove residue of Wash Solution.
13. Transfer the BP-10 96-Well Plate to a 96-well storage plate. Add 30-50 ul Elution Buffer to the BP-10 96-well plate; incubate at 50 oC for 2 minutes. Centrifuge at 6,000 rpm for 5 minutes.
14. Genomic DNA is ready for use or kept at – 20 oC.
15. 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.

From Paraffin Tissue

1. Excise 25~30mg paraffin tissue with a clean, sharp scalpel. And transfer to a Deep Well Collection Plate.
2. Add 1.2ml xylene (self-prepared by user) to Deep Well Collection Plate, Seal, and then vortex for 3 minutes. 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 supernatant completely.
7. Repeat washing step once from step 4 to 6.
8. Incubate at 37 oC for 10-15 minutes to remove residual ethanol.
9. Resuspend the sample in 200ul TE buffer, and continue immediately with Step 10.
10. Add 300 ul of ACL Solution (Animal Cell Lysis Solution) to Deep Well Collection Plate and add 20ul proteinase K, then seal it.
11. Incubate at 55oC until the tissue is completely lysed (usually 1-3 hours). Occasionally vortex.
12. Cool to room temperature. Vortex for 20 seconds and Centrifuge 12000rpm for 5 minutes.
13. Pipette 300ul of supernatant into an BP-10 96-Well Plate (if pellet not visible, repeat previous step) and add 300ul of AB Solution, Seal. Mix by occasionally inverting Plate, and keep for 2 minutes.
14. Centrifuge 6000rpm for 2 minutes and discard the flow-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. Spin at 6,000 rpm for 5 minutes to remove residual amount of Wash Solution.
18. Place the BP-10 96-Well Plate to 96-well storage plate. Add 30-50 ul Elution Buffer into the BP-10 96-Well Plate. Incubate the tube at 37 or 50 oC for 2 minutes. Incubate at 37 or 50 oC could increase recovery yield.
19. Spin at 8,000 rpm for 5 minutes to elute DNA from the column.
20. 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.

GDMIP-96-2 (96-well_genomic_kit-animal.doc, 65 kb)