Soil DNA extraction & WGA Amplification - Protocol

Genomic DNA from soil samples can be easily damaged by nucleases and contaminating debris resulting in low DNA yields. As a result, the researcher’s ability to perform downstream analysis may be compromised. After isolating DNA from the soil sample, follow GenomePlex Whole Genome Amplification Protocol

The UltraClean Soil Kit by MO Bio Laboratories Inc. is recommended for this procedure.

1. Add 0.5 gm of soil sample to the 2 ml Bead Solution tube provided.

2. Gently vortex to mix.

3. Add 60 μl of Solution 1 and invert several times.

4. Add 200 μl of Solution IRS (Inhibitor Removal Solution).

5. Vortex at maximum speed for 10 minutes.

6. Centrifuge at 10,000 × g for 30 seconds (be sure NOT to exceed 10,000 × g or the tubes may break).

7. Transfer the supernatant to a clean microcentrifuge tube (supernatant may contain some soil particles).

8. Add 250 μl of Solution S2 and vortex for 5 seconds.

9. Incubate at 4 °C for 5 minutes.

10. Centrifuge the tubes at 10,000 × g for 1 minute.

11. Transfer 450 μl of supernatant to a clean microcentrifuge tube.

12. Add 900 μl of Solution S3 to the supernatant and vortex for 5 seconds.

13. Load 700 μl of the solution onto a spin filter and centrifuge at

10,000 × g for 1 minute.

14. Discard the flow-through and add the remaining supernatant to the spin filter and centrifuge at 10,000 × g for 1 minute

15. Add 300 μl of Solution S4 and centrifuge for 30 seconds at 10,000 × g. Discard the flow-through.

16. Centrifuge for 1 minute.

17. Carefully place the spin filter into a clean microcentrifuge tube.

18. Add 50 μl of Solution S5 to the filter membrane.

19. Centrifuge for 30 seconds and discard the spin filter.

20. Store the eluted DNA at –20 °C or proceed to the amplification step.

Note: If using WGA2 there is no need to supply DNA polymerase as the enzyme is provided with the kit

WGA Amplification

Protocol for GenomePlex Whole Genome Amplification performed with GenomePlex Whole Genome Amplification Kit (WGA1) and/or GenomePlex Complete Whole Genome Amplification Kit (WGA2).

Fragmentation
Prepare DNA solution of 1 ng/ml from whole blood extraction protocol described above.

Add 1 μl of 10X Fragmentation Buffer to 10 ml DNA (1 ng/μl) in a PCR tube.

Place the tube in a thermal cycler at 95 °C for exactly 4 minutes. Note, the incubation is time sensitive and any deviation may alter results.

Immediately cool the sample on ice and centrifuge briefly.

Library Preparation
Add 2 μl of 1x Library Preparation Buffer.

Add 1 μl of Library Stabilization Solution.

Mix thoroughly and place in thermal cycler at 95 °C for 2 minutes.

Cool the sample on ice and centrifuge briefly.

Add 1 μl Library Preparation Enzyme, mix thoroughly, and centrifuge briefly.

Place sample in thermal cycler and incubate as follows:

    16 °C for 20 minutes
    24 °C for 20 minutes
    37 °C for 20 minutes
    75 °C for 5 minutes
    4 °C hold
Remove samples from thermal cycler and centrifuge briefly. Samples may be amplified immediately or stored at - 20 °C up to three days.

Amplification
Add the following reagents to the entire 15 μl reaction:

    7.5 μl 10x Amplification Master Mix
    47.5 μl Nuclease Free Water
    5.0 μl JumpStart Taq DNA Polymerase (12.5 units) for WGA1
    -or-
    5.0μml WGA DNA Polymerase for WGA2

Mix thoroughly, centrifuge briefly, and begin thermocycling:

    Initial Denaturation: 95 °C for 3 minutes
    Perform 14 cycles as follows:
    Denature: 95 °C for 15 seconds
    Anneal/Extend: 65 °C for 5 minutes

After cycling is complete, maintain the reactions at 4 °C or store at –20 °C until ready for analysis or purification.


Reamplification

1. Add 10 μl of 1 ng/μl WGA amplified DNA to a PCR tube or multiwell plate.
Note: It is necessary to clean up the WGA reaction to decrease possible bias in the reamplification. We recommend using the Sigma’s GenElute™ PCR Clean-Up Kit (Product Number NA1020) or standard purification methods that isolate single and double stranded DNA.

2. Create amplification mix. For each reamplification reaction, add the following to the WGA amplified DNA (step 1):

    47.5 μl of Nuclease-Free Water
    7.5 μl of 10X Amplification Master Mix
    5 μl of WGA DNA Polymerase

3. Vortex thoroughly, centrifuge briefly, and begin thermocycling. The following profile has been optimized for a PE 9700 or equivalent thermocycler:

    Initial Denaturation 95 °C for 3 minutes
    Perform 14 cycles as follows:
    Denature 94 °C for 15 seconds
    Anneal/Extend 65 °C for 5 minutes

After cycling is complete, maintain the reactions at 4 °C or store at –20 °C until ready for analysis or purification. The stability of WGA DNA is equivalent to genomic DNA stored under the same conditions.

Purification

Purification of Amplified Products performed with GenElute PCR Clean-Up Kit (NA1020)

1. Insert a GenElute Miniprep Binding Column (with a blue O-ring) into a provided collection tube, if not already assembled. Add 0.5 ml of the Column Preparation Solution to each miniprep column and centrifuge at 12,000 x g for 30 seconds to 1 minute. Discard the eluate.

Note: The Column Preparation Solution maximizes binding of the DNA to the membrane resulting in more consistent yields.

2. Add 5 volumes of Binding Solution to 1 volume of the PCR reaction and mix. For example, add 500 ml of Binding Solution to 100 ml of the PCR reaction. Transfer the solution into the binding column. Centrifuge the column at maximum speed (12,000 to 16,000 x g) for 1minute. Discard the eluate, but retain the collection tube.

3. Replace the binding column into the collection tube. Apply 0.5 ml of diluted Wash Solution to the column and centrifuge at maximum speed for 1minute. Discard the eluate, but retain the collection tube.
Note: Be sure to add ethanol to the Wash Solution Concentrate prior to first time use. See Preparation Instructions.

4. Replace the column into the collection tube. Centrifuge the column at maximum speed for 2minutes, without any additional wash solution, to remove excess ethanol. Discard any residual eluate as well as the collection tube.

5. Transfer the column to a fresh 2ml collection tube. Apply 50μl of Elution Solution or water to the center of each column. Incubate at room temperature for 1minute.
Note: When eluting with water, make sure that the pH of the water is between 5.5 and 8.5. Elution may also be performed using the Elution Solution diluted 10-fold with water.

6. To elute the DNA, centrifuge the column at maximum speed for 1 minute. The PCR amplification product is now present in the eluate and is ready for immediate use or storage at –20°C.

Quantification of Amplified Products

The amount of DNA amplified using GenomePlex Whole Genome Amplification kits can be detected with or without purification. For the highest quality samples of DNA, it is strongly recommended to purify the samples after amplification. The amplified products can be measured with the PicoGreen® dsDNA Quantitation Assay from Molecular Probes Inc. (#P-7589). Another method of detecting the amplified products is spectrophotometric absorption (OD260) on a NanoDrop® spectrophotometer. This instrument can measure absorbance on 1 μl of sample over a large dynamic range, from 2–3700 ng/

Application Data

soil1

Whole Genome Amplification Performed on a Soil Sample

GenomePlex WGA Amplified Sigma products are resolved on a 1.5% agarose gel. 5 μl of amplified product was loaded per well. The GenomePlex amplified products result in an average size of 400 bp. The smear pattern is source specific as shown on the gel.
Extraction of DNA from Soil
Lane 1—1 kb Ladder
Lane 2—Blood
Lane 3—Plant
Lane 4—Buccal Swab
Lane 5—Soil
Lane 6—Positive Control
Lane 7—1 kb Ladder

Materials

     
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