WGA5 Amplification

The flexibility of the GenomePlex® technology enables the use of compromised starting material, such as DNA isolated from FFPE tissue, for whole genome amplification. Whole genome amplification directly from formalin-fixed, paraffin-embedded (FFPE), frozen, RNAlater™-preserved or fresh tissue is now possible with our GenomePlex Tissue Whole Genome Amplification Kit (Product No. WGA5). The kit includes optimized reagents for tissue disruption and cell lysis eliminating the need for tedious organic extractions to remove excess paraffin or DNA purification prior to amplification. This rapid and straightforward method yields microgram quantities of genomic DNA from one milligram of tissue.

WGA5 Protocol
Lysis Fragmentation

1. Weigh out a 1 mg sample of fresh, frozen, RNAlater-preserved, or FFPE tissue into a PCR-ready vessel. See step 1 of Library Preparation for instructions on setting up a positive control reaction.
   Note: As little as 0.1 mg of tissue has been successfully used with this kit. If use of less than 1 mg of FFPE tissue is desired, it is recommended to start with 1 mg of tissue and then empirically determine the minimum amount of tissue that can be processed.
2. To the sample add 24 µL of CelLytic Y Lysis Solution (Product No. C8367) and 6 µL of Proteinase K Solution (Product No. P4850).
3. Place the tube in a thermal cycler at 60 °C for 60 minutes and then 99 °C for 4 minutes.

Library Preparation

1. Combine 9 µL of Nuclease-Free Water (Product No. W4765) and 1 µL of the tissue lysate from step 3 of Lysis and Fragmantation into a fresh PCR-ready vessel. For a positive control reaction, combine 7 µL of Nuclease-Free Water (Product No. W4765), 2 µL of Control Human Genomic DNA (Product No. D7192), and 1 µL of CelLytic Y Lysis Solution (Product No. C8367).
2. Add 2 µL of 1× Library Preparation Buffer (Product No. L7167).
3. Add 1 µL of Library Stabilization Solution (Product No. L7292).
4. Mix thoroughly and place in thermal cycler at 95 °C for 2 minutes.
5. Immediately cool the sample on ice, consolidate the sample by centrifugation, and replace on ice.
6. Add 1 µL of Library Preparation Enzyme (Product No. E0531), mix thoroughly, and centrifuge briefly.
7. Place sample in a 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
8. Remove samples from thermal cycler and centrifuge briefly. Samples may be amplified immediately or stored at –20 °C for three days.

Amplification

1. Add the following reagents to the entire 14 µl reaction:
   7.5 µl of 10× Amplification Master Mix
   48.5 µl of Nuclease-Free Water
   5.0 µl of WGA DNA Polymerase
2. Mix thoroughly, centrifuge briefly and begin thermocycling. The following profile has been optimized for a PE 9700 or equivalent thermal cycler:
   

   Initial Denaturation	95 °C for 2 minutes
   Perform 20 cycles as follows:
   Denature	94 °C for 15 seconds
   Anneal/Extend	65 °C for 4 minutes
   Hold	4 °C

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.