The Pinpoint™ Slide DNA Isolation System is an innovative product for the isolation of total DNA from targeted areas of fresh, frozen, and FFPE tissue sections. There is no need for expensive specialized equipment or computer software. Instead, the system combines innovative Pinpoint™ tissue sampling technology, Proteinase K digestion, and a one-step DNA extraction method for the isolation of DNA that is ideal for PCR, sequencing, etc.
The purpose of this procedure is to remove the selected tissue area from the slide.
Apply the Pinpoint Solution over the area you want to recover DNA on the prepared slide.
Use a pipette tip or a syringe to gently spread a small amount of Pinpoint Solution over the selected region.
Use about 0.5 µl of Pinpoint Solution per mm2 of tissue area or cover the selected area with a 0.5 mm
layer of Pinpoint Solution. It is OK to spread the Pinpoint Solution beyond area that you had selected. It is actually easier to define the exact tissue area for recovery in step 3, see below.
Dry the Pinpoint Solution completely at room temperature. It usually takes about 30 to 45 minutes.
The Pinpoint Solution will change into a thin blue film after completely drying; the underlying tissue cells are embedded in the film at this stage.
Remove the embedded tissue from the slide.
Use a clean and sharp knife to cut the exact area you want, then peel the area off the slide.
Transfer the film to a 0.5 ml tube. Generally, a minimal 1 mm2 tissue area with 10 µm thickness (about 200-400 cells depending on different tissue and cell density) is needed for successful gene amplification.
The size of each sample spot can vary from 1 to over 20 mm2 according to your needs.
Centrifuge the tubes briefly to bring the fragment to the tube bottom.
This one buffer extraction procedure will extract DNA from the recovered tissue.
Add 50 µl of Extraction Buffer and 5 µl Proteinase K to the tube containing the recovered tissue. Mix gently.
For multiple samples, Extraction Buffer and Proteinase K can be premixed, add 55 µl of this mixture to each sample.
Incubate the tubes at 55°C for 4 hours.
Heat the tubes at 95°C-98°C for 10 minutes then immediately put them on ice. This can be easily accomplished on a PCR machine.
Make sure that the temperature is above 95°C. Incomplete inactivation of Proteinase K can cause problems in the PCR reaction.
Vortex hard for 10-15 seconds. Centrifuge briefly. The DNA is ready for PCR analysis.
Attention: In most cases the DNA is suitable for PCR analysis at this step and there is no need to proceed to the following DNA Purification steps.
Use 4-8 µl of above DNA for each PCR reaction in 25-50 µl volume.
Sometimes tissue processing procedures, mainly fixing and staining processes, inadvertently add PCR inhibitors to the tissue and the DNA needs further purification to eliminate the inhibitors.
Please follow the DNA Purification procedure below to purify the DNA further.
Note: Add 14 ml 95-100% ethanol to the PP Wash Buffer Concentrate to make final PP Wash Buffer.
Add 100 µl Pinpoint Binding Buffer to the above Proteinase K treated DNA sample. Mix briefly.
Transfer the mixture to a Zymo-Spin I Column and place the column into a 2 ml Collection Tube.
Spin the column and tube at full speed in a microfuge for 10 seconds.
Add 150 µl PP Wash Buffer to the Zymo-Spin I Column and centrifuge at full speed for 10 seconds to wash.
Add another 150 µl PP Wash Buffer and centrifuge at full speed for one minute.
Transfer the column into a new 1.5 ml tube.
Add 10 µl water or TE Buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) directly to the membrane of Zymo Spin I Column.
Wait for 1 minute. Spin briefly for 10 seconds to elute the DNA.
The eluted DNA now can be used for PCR amplification or can be stored at -20°C for future uses. Use 2-4 µl of the purified DNA for each PCR reaction in 25-50 µl reaction volume.
For specific notes and additional information, please see the product protocol PDF.
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