DNA Isolation Flashcards
(19 cards)
After centrifuging buffy coat samples at 300 x g for 10 minutes at room temperature, what is the next step?
Obtain 200 μL of buffy coat and transfer to a new microcentrifuge tube
If the buffy coat volume is already 200 μL, what is added next?
Add 20 μL Proteinase K, quick vortex, and fast spin
After adding Proteinase K, what enzyme is added and for how long is the sample incubated?
Add 20 μL RNase and incubate at room temperature for 2 minutes
What buffer is added after RNase treatment, and what is the next step?
200 μL PureLink® Genomic Lysis/Binding Buffer, vortex to homogenize, and perform a fast spin
How long and at what temperature do you incubate after adding the lysis/binding buffer?
Incubate at 55°C for 10 minutes
What do you do after incubating for 10 minutes?
Add 200 μL absolute ethanol, vortex to homogenize, and perform a fast spin
After adding ethanol, where do you transfer the lysate?
Transfer the lysate to a PureLink® Spin Column Collection Tube
What is done after centrifugation of lysate?
Discard the collection tube and place the spin column into a clean collection tube
How long do you centrifuge the lysate in the spin column?
Centrifuge at maximum speed for 3 minutes at room temperature. If needed, repeat for 1 minute until no clot is visible
What is added for the first wash step, and how long do you centrifuge?
Add 500 μL PureLink® Wash Buffer 1 (prepared with ethanol) and centrifuge at maximum speed for 1 minute
What is discarded after the first wash step, and what is added for the second wash?
Discard the collection tube, place the spin column into a new collection tube, and add 500 μL PureLink® Wash Buffer 2 (Ethanol)
How long do you centrifuge for the second wash step?
Centrifuge at maximum speed for 3 minutes
After washing, where do you place the spin column?
Place the spin column into a sterile 1.5 mL microcentrifuge tube
What should you do if the buffy coat volume is less than 200 μL?
Add PBS to make the final volume 200 μL and perform a fast spin
What do you add to the spin column for elution, and how long do you incubate?
Add 50 μL PureLink® Genomic Elution Buffer and incubate at room temperature for 1 minute
How long do you centrifuge after adding the elution buffer to obtain the DNA eluate?
Centrifuge at maximum speed for 1 minute at room temperature
What should you do to recover more DNA after the first elution step?
Perform a second elution step using the same elution buffer and centrifuge at maximum speed for 1.5 minutes
How can you increase DNA yield during the second elution?
Add 50 μL in the column with a new 1.5 mL microcentrifuge tube and centrifuge at maximum speed for 1 minute
At what temperature should the DNA eluates be stored after the isolation process?
Store DNA eluates in a freezer at -20°C
