Plant DNA extraction & WGA Amplification-protocol

Extracting DNA from plant tissue is a complicated process due to the tough cell wall that surrounds most plant cells. Genomic DNA from plant material can be damaged during the extraction process, resulting in low yields of high quality genomic material. As a result,the researcher’s ability to perform downstream analysis is challenged. GenomePlex Whole Genome Amplification has been used to amplify genomic DNA from soybean, corn, tomato, purple coneflower, and ginseng.

It is recommended to use the Sigma GenElute Plant Genomic DNA Miniprep Kit (G2N10) for this procedure.

1. Grind approximately 50 mg leaf punch into a fine powder with liquid nitrogen. Keep the sample on ice for immediate use or freeze at –70 °C.

2. Add 350 μl of Lysis Solution (Part A) and 50 μl of Lysis Solution (Part B) and thoroughly mix by vortexing. A white precipitate will form upon the addition of Lysis Solution Part B.

3. Incubate the mixture at 65 °C for 10 minutes with occasional inversion to dissolve the precipitate.

4. Add 130 μl of Precipitation Solution, mix by inversion, and place the sample on ice for 5 minutes.

5. Centrifuge at maximum speed (12,000–16,000 × g) for 5 minutes to pellet the cellular debris, proteins and polysaccharides.

6. Carefully pipette the supernatant onto a GenElute Filtration Column (blue insert with a 2 ml collection tube).

7. Centrifuge at maximum speed for 1 minute. Discard the Filtration Column and retain the collection tube.

8. Add 700 μl of Binding Solution directly to the flow-through (liquid from step 7). Mix thoroughly by inversion.

9. Insert the GenElute Miniprep Binding Column (red o-ring) into the provided microcentrifuge tube.

10. Add 500 μl of the Column Preparation Solution to each Miniprep Column and centrifuge at 12,000 × g for 1 minute. Discard the flow-through liquid.

11. Pipette 700 μl flow-through (from step 8) onto the Miniprep Column prepared in the previous step.

12. Centrifuge at maximum speed for 1 minute and discard the flowthrough.

13. Apply the remaining lysate (from step 8) and repeat centrifugation for 1 minute at maximum speed and discard the flow-through.

14. Place the Binding Column in a fresh 2 ml collection tube and apply 500 μl diluted Wash Solution to the column (be sure to add ethanol to the Wash Solution Concentrate prior to first time use).

15. Centrifuge at maximum speed for 1 minute. Discard flow-through and retain the collection tube.

16. Add another 500 μl of diluted Wash Solution to the column and centrifuge at maximum speed for 3 minutes to dry the column.

17. Transfer the binding column to a fresh 2 ml collection tube.

18. Apply 100 μl of pre-warmed (65 °C) Elution Solution to the column and centrifuge at maximum speed for 1 minute. Repeat the elution.

19. Store the eluted DNA at –20 °C or proceed to the amplification step using WGA
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 Complete Whole Genome Amplification Kit (WGA2).

Fragmentation

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

Add 1 µl of 10X Fragmentation Buffer to 10 µl 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 ml 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-
    5.0 µl 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.
Replace the binding column into the collection tube.

3. 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 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/

Plant tissue

GenomePlex® Whole Genome Amplification Performed on Plant Samples

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 varies by source as shown on the gel. Products amplified using GenomePlex
technology are specific as evidenced by the lack of signal in the negative control (lane 3).
Lane 1—1 kb Marker
Lane 2—Positive Control
Lane 3—Negative Control
Lane 4—Corn
Lane 5—Ginseng
Lane 6—Purple Cornflower
Lane 7—Tomato
Lane 8—Soybean

Required Products

G2N10
E7023
W4502
WGA1
WGA2
WGA3
T9661
D9307

Materials to be Supplied by the User

Plant Samples
Microcentrifuge (with rotor for 2 ml tubes)
55 °C water bath or heat block

Materials

     
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