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E.Z.N.A.TM Plant DNA Maxi Protocol For Dry Specimens

互联网2013-11-13

1065

实验试剂

1. Equilibrate Elution Buffer or 10 mM Tris pH 9.0 at 65°C.

2. Isopropyl alcohol (isopropanol)

3. Absolute (96%-100%) ethanol

实验设备

1. High speed centrifuge capable of at least 10,000 x g

2. Centrifuge with swinging-bucket rotor at room temperature capable of 4000xg.

3. Nuclease-free 50 ml high speed centrifuge tubes (such as Nelgen polycarbonate tube Cat#3118-0050) and 50ml centrifuge tubes capable of 4000 x g centrifugation.

4. Waterbath equilibrated to 65°C

实验步骤

1. To up to 500 mg powdered dry tissue add 16 ml Buffer P1. Vortex vigorously to mix. Make sure to disperse all clumps.

2. Incubate at 65°C for 30-60 min. Mix sample by vortexing during incubation.

3. Add 2.8 ml Buffer P2 and vortex to mix. Incubate on ice for 10 minutes. Centrifuge at 10,000 x g for 15 min.

4. Carefully aspirate supernatant to a new 50 ml hi-speed centrifuge tube making sure not to disturb the pellet or transfer any debris. Add 0.7 volume isopropanol and vortex to precipitate DNA. This step will remove much of the polysaccharide content and improves spin-column performance by increasing DNA binding capacity (and hence yield) in the steps that follow.

TIP: In most cases 16 ml supernatant can easily be removed. This will require 11.2 ml isopropanol. Note that depending on the sample, the volume of supernatant may vary.

5. Centrifuge at 10,000 x g for 15 min to pellet DNA. Longer centrifugation does

6. Carefully aspirate or decant the supernatant and discard making sure not to dislodge the DNA pellet. Invert the centrifuge tube on a paper towel for 5 min to allow residual liquid to drain. Do not over dry the DNA pellet.

7. Add 4 ml of sterile deionized water pre-heated to 65°C and 50ul RNase A (Supplied). Vortex to resuspend the pellet.. Incubate at 65°C for 10 minutes. It may be necessary to remove un-dissolved material by centrifuging at 10,000 x g for 5 minutes.

TIP: While incubating at 65°C to dissolve the DNA, label and place the required number of HiBind® DNA columns in 50 ml collection tubes (supplied).

8. Adjust binding conditions of the sample by adding 2 ml Buffer P3 followed by 4 ml absolute ethanol and vortex to obtain a homogeneous mixture. A precipitate may form upon addition of ethanol; it will not interfere with DNA isolation.

9. Apply the entire sample (including any precipitate that may have formed) to an HiBind® DNA Maxi-column placed in a 50 ml collection tube (supplied). Centrifuge the column at 3,500 x g for 15 min to bind DNA. Discard both the 50 ml collection tube and the flow-through liquid.

10. Transfer column to a second 50ml collection tube (not supplied) and wash by adding 10 ml HB Buffer. Centrifuge at 4,000 x g for 3 min and discard the flow-through liquid. Reuse the collection tube in Step 11 below.

11. Add 15 ml DNA Wash Buffer to the column. Centrifuge at 4,000 x g for 3 min. Discard flow-through and reuse 50 ml collection tube in Step 12.

NOTE: DNA Wash Buffer Concentrate must be diluted with absolute (96%-100%) ethanol prior to use. Follow directions on label.

12. Wash the column with 15 ml DNA Wash Buffer to the column by centrifuge at 4000 x g for 3 min. Discard the floe through and reuse the 50 ml collection tube for set 13.

13. Centrifuge empty column 20 min at 4000x g to dry. This step is critical for removing residual ethanol that may interfere with downstream applications.

Note: When a vacuum oven is available, place the maxi column into a vacuum oven which is preset at 60°C for 10-15 minutes. This will ensure that the column can be completely dried before elution.

14. Transfer column to a clean 50 ml tube. Apply 2ml Elution Buffer or prewarmed to 65°C and incubate at room temperature for 5 min. Centrifuge at 4,000 x g for 10 min to elute DNA. Smaller volumes will significantly increase DNA concentration but give lower yields. Use of more than 4ml buffer for elution for elution is not recommended.

15. Repeat Step 14 with an additional 2 ml Elution buffer. This may be performed using another 1.5 ml tube to maintain a higher DNA concentration in the first eluate. To increase DNA concentration add buffer and incubate the column at 60°C -70°C for 5 min before elution.

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