- Materials
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- >300 cells in 0.5 µl nuclease-free water, or 1-10 ng DNA in 1 µl nuclease-free water
- Qiagen REPLI-g UltraFast Mini Kit
- Fragmentation Mix (FRA)
- Rapid Adapter (RAP)
- Consumables
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- Nuclease-free water (e.g. ThermoFisher, AM9937)
- 1.5 ml Eppendorf DNA LoBind tubes
- 0.2 ml thin-walled PCR tubes
- Equipment
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- Thermal cycler
- Ice bucket with ice
- Optional equipment
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- Qubit fluorometer (or equivalent for QC check)
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Input recommendation
The REPLI-g UltraFast Mini Kit suggests an input of either 300 cells‡, or 1-10 ng‡‡ of gDNA. The method describing how to perform whole genome amplification differs, depending on sample type (cells or DNA), and details for each are provided. Please also refer to manufacturer's guides for further details.
Notes:
‡ Although cells can be used in the REPLI-g kit, the chemical lysis step may not be sufficient to release the DNA from all cell types. Additional steps to ensure all cells are lysed may be required, depending on sample type.
‡‡ We have found that it is possible to reduce the input amount to, for example, 10 pg of gDNA. However, lowering the input may require the time of the amplification step to be increased. We recommend amplifying until the DNA concentration is >80 ng/μl (quantification performed using the Qubit dsDNA BR Assay Kit). -
If starting with whole cells, follow the instructions below.
Qiagen recommends that cells should be at a concentration of >600 cells/μl (please refer to manufacturer's guide for more information). If lower numbers of cells are used, please increase the amplification time until the concentration of amplified DNA is >80 ng/μl (quantification performed using the Qubit dsDNA BR Assay Kit).
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In a 0.2 ml thin-walled PCR tube, mix together in the following order:
Reagent Volume Cell sample 0.5 µl Phosphate buffered saline (PBS) 1 µl Reagent D2 from the REPLI-g UltraFast Mini Kit 1.5 µl Total 3 µl -
Incubate the reaction for 5 minutes at room temperature.
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Add 1.5 µl Stop Reagent.
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In a 0.2 ml thin-walled PCR tube, mix together in the following order:
Reagent Volume REPLI-g UltraFast Reaction Buffer 15 µl REPLI-g UltraFast Reaction Polymerase 1 µl Total 16 µl -
Ensure the components are thoroughly mixed by pipetting, and spin down.
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Add the lysed cells from the previous step to the tube.
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Ensure the components are thoroughly mixed by pipetting, and spin down.
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Incubate the reaction at 30° C until the DNA concentration is > 80 ng/µl. Quantification should be performed using the Qubit dsDNA BR Assay Kit.
Typically this reaction would take ~2 hours, depending on the cell concentration and quality of the starting mateiral.
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If starting with purified DNA, follow the instructions below.
Qiagen recommends that 1-10 ng of gDNA, in a volume of 1 μl, should be put into the reaction (please refer to manufacturer's guide for more information). We have found that it is possible to reduce the input amount to 10 pg of gDNA. However, lowering the input may require a longer amplification time. We recommend amplifying until the DNA concentration is >80 ng/μl (quantification performed using the Qubit dsDNA BR Assay Kit).
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Put the DNA in 1 μl into a clean tube.
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Add 1 μl Buffer D1 to the tube. Mix by vortexing and spin down.
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Incubate for 3 minutes at room temperature.
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Add 2 μl Buffer N1 to the tube. Mix by vortexing and spin down.
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In a 0.2 ml thin-walled PCR tube, mix together in the following order:
Reagent Volume REPLI-g UltraFast Reaction Buffer 15 µl REPLI-g UltraFast Reaction Polymerase 1 µl Total 16 µl -
Ensure the components are thoroughly mixed by pipetting, and spin down.
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Add the DNA from the previous step to the tube.
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Ensure the components are thoroughly mixed by pipetting, and spin down.
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Incubate the reaction at 30° C until the DNA concentration is > 80 ng/µl. Quantification should be performed using the Qubit dsDNA BR Assay Kit.
Typically this reaction would take ~2 hours, depending on the cell concentration and quality of the starting mateiral.
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Regardless of input material, continue with the protocol as follows:
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In a 0.2 ml thin-walled PCR tube, mix the following:
Reagent Volume Amplified DNA 2.5 µl Nuclease-free water 5 µl Fragmentation Mix (FRA) 2.5 µl Total 10 µl -
Ensure the components are thoroughly mixed by pipetting, and spin down.
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Incubate the tube at 30°C for 1 minute and then at 80°C for 1 minute. Briefly put the tube on ice to cool it down.
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Add 1 μl RAP to the 10 μl amplified DNA library.
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Ensure the components are thoroughly mixed by pipetting, and spin down.
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Incubate the reaction for 5 minutes at room temperature.