TITLE: Invitrogen PureLink® Genomic DNA Mini Kit
AUTHORS: Shlomi Abuchatzera Green

[STEPS]
?. [Preparing lysates: Mammalian cells, tissues]
Set a water bath or heat block at
55 °C
?. [Preparing lysates: Mammalian cells, tissues]
For adherent cells (≤5 × 106 cells): remove the growth medium from the culture plate and harvest cells by trypisinization or a method of choice.For suspension cells (≤5 × 106 cells): harvest cells and centrifuge the cells at250 × g for 5 minutes to pellet cells. Remove the growth medium.
?. [Preparing lysates: Mammalian cells, tissues]
Resuspend the cells from Step 2 in  PBS.
200 µl
?. [Preparing lysates: Mammalian cells, tissues]
Add  Proteinase K (supplied with the kit, stored in ) to the sample.
20 µl
4 °C
?. [Preparing lysates: Mammalian cells, tissues]
Add  RNase A (supplied with the kit, stored in ) to the sample, mix well by brief vortexing, and incubate at room temperature for  2 minutes .
20 µl
4 °C
?. [Preparing lysates: Mammalian cells, tissues]
Add  PureLink® Genomic Lysis/Binding Buffer and mix well by vortexing to obtain a homogenous solution.
200 µl
?. [Preparing lysates: Mammalian cells, tissues]
Incubate at  for 10 minutes  to promote protein digestion.
55 °C
?. [Preparing lysates: Mammalian cells, tissues]
Add 96–100% ethanol to the lysate. Mix well by vortexing for 5 seconds to yield a homogenous solution.
200 µl
?. [Binding DNA]
Remove a PureLink® Spin Column in a Collection Tube from the package.
?. [Binding DNA]
Add the lysate (~640 μL) to the PureLink® Spin Column.
?. [Binding DNA]
Centrifuge the column at 10,000 × g for 1 minute at room temperature.
Centrifuge: 10000 34, 1 min
If you are processing >200 μL starting material such as blood, buccalswabs, or Oragene™ preserved saliva, you need to perform multiple loadingof the lysate by transferring any remaining lysate to the same PureLink® SpinColumn (above) and centrifuge at 10,000 × g for 1 minute.
?. [Binding DNA]
Discard the collection tube and place the spin column into a clean PureLink® Collection Tube supplied with the kit.
?. [Washing DNA]
Add  Wash Buffer 1 prepared with ethanol to the column.
500 µl
?. [Washing DNA]
Centrifuge column at room temperature at 10,000 × g for 1 minute.
Centrifuge: 10000 34, 1 min
?. [Washing DNA]
Discard the collection tube and place the spin column into a clean PureLink® collection tube supplied with the kit.
?. [Washing DNA]
Add  Wash Buffer 2 prepared with ethanol to the column.
500 µl
?. [Washing DNA]
Centrifuge the column at maximum speed for 3 minutes at room temperature.
Centrifuge: 16000 34, 3 min
?. [Washing DNA]
Discard collection tube.
?. [Eluting DNA]
Place the spin column in a sterile 1.5-mL microcentrifuge tube.
?. [Eluting DNA]
Add 25–200 μL of PureLink® Genomic Elution Buffer to the column.
?. [Eluting DNA]
Incubate at room temperature for 1 minute.
0 Room temperature
?. [Eluting DNA]
To recover more DNA, perform a second elution step using the same elution buffer volume as first elution in another sterile, 1.5-mL microcentrifuge tube.
?. [Eluting DNA]
Centrifuge the column at maximum speed for 1.5 minutes at room temperature.
Centrifuge: 16000 34, 90 s
?. [Eluting DNA]
The tube contains purified DNA. Remove and discard the column.
?. [Eluting DNA]
Centrifuge the column at maximum speed for 1 minute at room temperature.
Centrifuge: 16000 34, 1 min
The tube now contains purified genomic DNA.
?. [Storing DNA]
Store the purified DNA at –20°C or use DNA for the desired downstream application.
?. [Storing DNA]
For long-term storage, store the purified DNA in PureLink® Genomic Elution Buffer at –20°C as DNA stored in water is subject to acid hydrolysis.
?. [Storing DNA]
To avoid repeated freezing and thawing of DNA, store the purified DNA at 4°C for immediate use or aliquot the DNA and store at –20°C for long-term storage.