Amplification Protocol for PicoPLEX WGA Kit

Protocol

1. Combine the following Amplification Cocktail components and mix well:

   AMPLIFICATION COCKTAIL	VOLUME PER 5 SAMPLES
   Amplification Reaction Mix	125 μl
   Amplification Enzyme	4 μl
   Nuclease-Free Water	171 μl
   Total volume	300 μl

   Note: Sample amplification efficiency may be analyzed using a real-time qPCR instrument by adding SYBR Green I Dye at 0.125X final concentration in the Amplification Cocktail. Some instruments require additional dyes for signal normalization.
   
   
2. Mix 60 μl of the freshly prepared Amplification Cocktail with the 15 μl pre-amp incubation product and mix gently by pipet.
   
   
3. Amplify sample according to the thermal cycler program below:
   

   CYCLES	TEMP	TIME
   1	95°C	2 minutes
   14	95°C	15 seconds
   	65°C	1 minute
   	75°C	1 minute

   
   Note: 14 cycles are recommended based on testing performed with flow-sorted cultured cells. Some cell types may require up to 16 cycles to obtain maximal yields.
   
   
4. Immediately store the amplified product at -20°C or purify.
   
   Note: Many applications require purifying and quantifying WGA products before use. Products amplified using this kit can be purified with spin columns or filter plates.
   
   
5. Quantitate purified amplification products by UV absorbance (1 OD₂₆₀ = 50 μg/ml). Store the purified amplification product at -20°C.
   
   Note: PicoGreen™ or other double-stranded specific methods for DNA measurement will not give reliable product concentrations. The amplified material will be a mixture of predominantly double-stranded material with some single-stranded material present.